Modified iscb nucleases, modified guide rnas, and uses thereof

Abstract

Disclosed herein are novel polypeptides having nuclease activity. The polypeptides can effect modifications at defined locations within target nucleic acids, e.g., DNA and RNA. Also disclosed herein modified guide RNAs. The polypeptides and guide RNAs are useful in compositions, systems and methods, to make site specific alterations of target nucleic acid sequences.

Description

Attorney Docket No.: 098791-000102WOPT MODIFIED ISCB NUCLEASES, MODIFIED GUIDE RNAS, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 456,332 filed March 31, 2023, and U.S. Provisional Application No. 63 / 456,322 filed March 31, 2023, the contents of each of which are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The disclosure relates generally to DNA and RNA targeting enzymes, systems, and compositions that are useful for modifying target nucleic acid sequences. BACKGROUND

[0003] EboIscB (insertion sequences Cas9-like OrfB) is an RNA-guided endonuclease with measurable activity in human cells. However, the EboIscB in human cells is low. Further, to improve the utility of EboIscB as gene editing tool in human cells, guide RNAs that are shorter and have higher efficacy are needed. Thus, there remains a need in the art for RNA-guided endonucleases and guide RNAs having improved and enhanced activity, e.g., in human cells. This disclosure answers these needs. SUMMARY

[0004] Described herein are DNA and RNA targeting enzymes and systems, that are useful for modifying target nucleic acid sequences. Accordingly, provided herein is an engineered or non- naturally occurring DNA and RNA targeting enzymes and systems that include an engineered guide nucleic acid comprising a guide sequence (i.e., a spacer), where the guide sequence is capable of hybridizing with a target sequence of a target nucleic acid molecule.

[0005] Accordingly in one aspect provided herein is an IscB protein (also referred to as a protein effector polypeptide herein) comprising an amino acid sequence that is at least 80% identical to the amino SEQ ID NO: 2 (pEDIT071) or SEQ ID NO: 8 (pEdit071+NLS). For example, the IscB protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 2. In some embodiments, the IscB protein comprises an amino acid sequence of SEQ ID NO: 2, and wherein the amino acid sequence of the IscB protein comprises a mutation at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine positions selected independently from 18, 34, 36, 37, 60, 66, 85, 97, 99, 100, 101, 104, 109, 112, 118, 136, 137, 155, 156, 157, 166, 168, 190, 204, 204, 205, 206, 208, 212, 214, 216, 218, 219, 229, 229, 230, 233, 233, 234, 234, 238, 239, 241, 242, 245, 246, 248, 252, 254, 255, 256, 257, 258, 279, 280, 291, 293, 298, 303,Attorney Docket No.: 098791-000102WOPT 304, 306, 307, 312, 319, 323, 328, 329, 331, 332, 338, 341, 353, 360, 368, 372, 376, 377, 378, 379, 380, 381, 382, 384, 385, 386, 388, 390, 391, 393, 405, 406, 408, 409, 410, 412, 413, 416, 417, 418, 419, 421, 421, 423, 425, 437, 439, 455, 462, 463, 478, 479, 533, 536, 564 and 565 of the amino acid sequence of SEQ ID NO: 2 (pEDIT071). Optionally, if the IscB comprises a single mutation, the mutation is not Q60I, N66H, I85R, I85K, E137R, P405R, E409R, D413K, or combination thereof.

[0006] In some embodiments, the IscB protein comprises an amino acid sequence of SEQ ID NO: 8, and wherein the amino acid sequence of the IscB protein comprises a mutation at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine positions selected independently from 30, 46, 48, 49, 72, 78, 97, 109, 111, 112, 113, 116, 121, 124, 130, 148, 149, 167, 168, 169, 178, 180, 202, 216, 216, 217, 218, 220, 224, 226, 228, 230, 231, 241, 241, 242, 245, 245, 246, 246, 250, 251, 253, 254, 257, 258, 260, 264, 266, 267, 268, 269, 270, 291, 292, 303, 305, 310, 315, 316, 318, 319, 324, 331, 335, 340, 341, 343, 344, 350, 353, 365, 372, 380, 384, 388, 389, 390, 391, 392, 393, 394, 396, 397, 398, 400, 402, 403, 405, 417, 418, 420, 421, 422, 424, 425, 428, 429, 430, 431, 433, 433, 435, 437, 449, 451, 467, 474, 475, 490, 491, 545, 548, 576, and 577 of the amino acid sequence of SEQ ID NO: 8 (pEDIT071 + NLS). Optionally, if the IscB comprises a single mutation, the mutation is not Q72I, H78H, I97R, I97K, E149R, P417R, E421R, D425K, or combination thereof.

[0007] In some embodiments, the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected independently from the group consisting of variant C18R, A34R, D36R, T37R, Q60I, N66H, I85X, I85X, M99X, A101X, A104R, E109X, A112K, R118K, S136X, E137R, I155X, L156X, T157X, T166X, L168K, L190K, G204X, N205X, I206K, K208R, K212R, K214R, N216R, K218R, D219X, A229X, K230R, L233X, P234X, P238X, F239X, K241R, E242X, A245X, E246X, E248R, T252X, E254X, K255R, S256X, T257X, F258X, S279R, G280R, L291X, K293R, Q298K, E303X, M304K, K306R, R307K, C312X, T319K, E323X, E328R, Q329R, D331R, G332R, G338R, S341R, R353K, A360X, K368R, L372R, D376R, A377R, D378R, T379R, A380R, E381R, S382R, T384R, E385R, M386R, T388R, L390X, M391X, K393R, P405K, K406R, V408R, E409X, T410R, A412R, D413R, P416R, G417R, H418K, F419R, V421X, N423R, W425K, E437R, D439X, E455R, E462R, P463R, I533K, T536R, H478X, H479X, I564X, and T565X of the amino acid sequence SEQ ID NO: 2, wherein X is any amino acid. Optionally, if the IscB comprises a single mutation, the mutation is not Q60I, N66H, I85R, I85K, E137R, P405R, E409R, D413K, or combination thereof.

[0008] In some embodiments, the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected independently from the group consisting of variant C30R, A46R, D48R, T49R, Q72I, N78H, I97X, I109X, M111X, A1113X, A116R, E121X, A124K, R130K, S148X, E149R, I167X, L168X, T169X, T178X, L180K, L202K, G216X, N217X, I218K, K2220R, K224R, K2226R, N228R,K230R, D231X, A241X, K242R, L245X, P246X, P250X, F251X, K253R, E254X, A257X, E258X, E260R, T264X, E266X, K267R, S268X, T269X, F270X, S291R, G292R, L303X, K2305R, Q310K, E315X, M316K, K318R, R319K, C324X, T331K, E335X, E340R, Q343R, G344R, G350R, S353R, R365K, A372X, K380R, L384R, D388R, A389R, D390R, T391R, A392R, E393R, S394R, T396R, E397R, M398R, T400R, L402X, M403X, K3405R, P417K, K418R, V420R, E421X, T422R, A424R, D425R, P428R, G429R, H430K, F431R, V433X, N435R, W437K, E449R, D451X, E467R, E474R, P475R, I545K, T548R, H490X, H491X, I576X, and T577X of the amino acid sequence SEQ ID NO: 8, wherein X is any amino acid. Optionally, if the IscB comprises a single mutation, the mutation is not Q72I, H78H, I97R, I97K, E149R, P417R, E421R, D425K, or combination thereof

[0009] In some embodiments, the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected independently from the group consisting of C18R, A34R, D36R, T37R, Q60I, N66H, I85R, I85K, D97R, D97K, M99R, M99K, A100K, A101R, A101K, A104R, E109R, E109K, A112K, R118K, S136R, S136K, E137R, E137K, I155R, I155K, L156R, L156K, T157R, T157K, T166R, T166K, L168K, L190K, G204R, G204K, N205R, N205K, I206K, K208R, K212R, K214R, N216R, K218R, D219R, D219K, A229R, A229K, K230R, L233R, L233K, P234R, P234K, P238R, P238K, F239R, F239K, K241R, E242R, E242K, A245R, A245K, E246R, E246K, E248R, T252R, T252K, E254R, E254K, K255R, S256R, S256K, T257R, T257K, F258R, F258K, S279R, G280R, L291R, L291K, K293R, Q298K, E303R, E303K, M304K, K306R, R307K, C312R, C312K, T319K, E323R, E323K, E328R, Q329R, D331R, G332R, G338R, S341R, R353K, A360R, A360K, K368R, L372R, D376R, A377R, D378R, T379R, A380R, E381R, S382R, T384R, E385R, M386R, T388R, L390R, L390K, M391R, M391K, K393R, P405K, K406R, V408R, E409K, E409R, T410R, A412R, D413R, D413K, P416R, G417R, H418K, F419R, V421R, V421K, N423R, W425K, E437R, D439R, D439K, E455R, E462R, P463R, I533K, T536R, H478N, H479N, I564K, I564H, I564N, I564Q, I564C, I564M, I564T, I564D, T565R, T565K, T565H, T565C, T565D, T565G, H478C, H478P, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2. Optionally, if the IscB comprises a single mutation, the mutation is not Q60I, N66H, I85R, I85K, E137R, P405R, E409R, D413K, or combination thereof.

[0010] In some embodiments, the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected independently from the group consisting of C30R, A46R, D46R, T49R, Q72I, N78H, I97R, I97K, D109R, D109K, Ml HR, Mi l IK, A112K, A113R, A113K, A116R, E121R, E121K, A124K, R130K, S148R, S148K, E149R, E149K, I167R, I167K, L168R, L168K, T169R, T169K, T178R, T178K, L180K, L1202K, G216R, G216K, N217R, N217K, I218K, K220R, K224R, K2226R, N228R, K230R, D231R, D231K, A241R, A241K, K242R, L245R, L245K, P246R, P246K, P250R, P250K, F251R, F251K, K253R, E254R, E2454K, A257R, A257K, E258R, E2458K, E260R, T264R, T264K, E266R, E266K, K267R, S268R, S268K, T2569R, T2569K, F270R, F270K, S291R,G292R, L303R, L303K, K305R, Q310K, E315R, E315K, M316K, K318R, R319K, C324R, C324K, T331K, E335R, E335K, E340R, Q341R, D343R, G344R, G350R, S353R, R365K, A372R, A372K, K380R, L384R, D388R, A389R, D390R, T391R, A392R, E393R, S394R, T396R, E397R, M398R, T400R, L402R, L402K, M4031R, M403K, K405R, P417K, K419R, V420R, E421K, E421R, T422R, A424R, D425R, D425K, P428R, G429R, H430K, F431R, V433R, V433K, N435R, W437K, E449R, D451R, D451K, E4567R, E474R, P475R, I545K, T548R, H490N, H491N, I576K, I576H, I576N, I576Q, I576C, I576M, I576T, I5676D, T577R, T577K, T577H, T577C, T577D, T577G, H490C, H490P, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 8. Optionally, if the IscB comprises a single mutation, the mutation is not Q72I, H78H, I97R, I97K, E149R, P417R, E421R, D425K, or combination thereof.

[0011] In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H478X, H479N, I564X, T565X, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2, wherein X is any amino acid. In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H478N, H479N, I564K, I564H, I564N, I564Q, I564C, I564M, I564T, I564D, T565R, T565K, T565H, T565C, T565D, T565G, H478C, H478P, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2.

[0012] In some embodiments, the IscB protein comprises an amino acid sequence comprising a mutation at position 478 and / or 479 of the amino acid sequence provided in SEQ ID NO: 2. In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutation H478N, H479N, H478C and / or H478P in the amino acid sequence provided in SEQ ID NO: 2.

[0013] In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E409R, D413K, 533K in SEQ ID NO: 2. In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E409R, D413K, and 533K, and at least one other mutation at a position listed in Table A in SEQ ID NO: 2. In some further embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E409R, D413K and 533K, and at least one, at least two, at least three, or four of the mutation E137K, N66H, A100K, and / or T536R in SEQ ID NO: 2. For example, the IscB protein comprises an amino acid sequence comprising the mutations E409R, D413K, 533K, E137K, N66H, A100K, and T536R in SEQ ID NO: 2.

[0014] In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E409R, D413K, and 533K; at least one, at least two, at least three, or four of the mutation E137K, N66H, A100K, and / or T536R; and at least one other mutation listed in Table A in SEQ ID NO: 2

[0015] In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E409R, D413K, 533K E137K, N66H, A100K and T536R, and at least one other mutation listed in Table A in SEQ ID NO: 2.

[0016] In some embodiments, the IscB protein comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, at least five, at least six or seven of the mutations E409R, D413K, 533K, E137K, N66H, A100K, and / or T536R, and at least one other mutation listed in Table A in SEQ ID NO: 2. For example, the IscB protein comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, at least five, at least six or seven of the mutations E409R, D413K, 533K, E137K, N66H, A100K, and / or T536R, and at least one other mutation listed in Table A in SEQ ID NO: 2, and where the at least one other mutation is not E409R, D413K, 533K, E137K, N66H, A100K, and / or T536R.

[0017] In some embodiments, the IscB protein one, two, three or four of the mutations E137K, N66H, A100K, and / or T536R in SEQ ID NO: 2. For example, the IscB protein one, two, three or four of the mutations E137K, N66H, A100K, and / or T536R, and at least one other mutation listed in Table A in SEQ ID NO: 2.

[0018] In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H490X, H49 IN, I576X, T577X, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 7, wherein X is any amino acid. In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H490N, H491N, I576K, I576H, I576N, I576Q, I576C, I576M, I576T, I576D, T577R, T577K, T577H, T577C, T577D, T577G, H490C, H490P, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 7.

[0019] In some embodiments, the IscB protein comprises an amino acid sequence comprising a mutation at position 490 and / or 491 of the amino acid sequence provided in SEQ ID NO: 7. In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutation H490N, H491N, H490C and / or H490P in the amino acid sequence provided in SEQ ID NO: 7.

[0020] In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E421R, D425K, 545K in SEQ ID NO: 7. In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E421R, D425K, and 545K, and at least one other mutation at a position listed in Table A2 in SEQ ID NO: 7. In some further embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E421R, D425K and 545K, and at least one, at least two, at least three, or four of the mutation E149K, N78H, Al 12K, and / or T548R in SEQ ID NO: 7. For example, the IscB protein comprises an amino acid sequence comprising the mutations E421R, D425K, 545K, E149K, N78H, Al 12K, and T548R in SEQ ID NO: 7.

[0021] In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E421R, D425K, and 545K; at least one, at least two, at least three, or four of the mutation E149K, N78H, Al 12K, and / or T548R; and at least one other mutation listed in Table A2 in SEQ ID NO: 7

[0022] In some embodiments, the IscB protein comprises an amino acid sequence comprising the mutations E421R, D425K, 545K E149K, N78H, A112K and T548R, and at least one other mutation listed in Table A2 in SEQ ID NO: 7.

[0023] In some embodiments, the IscB protein comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, at least five, at least six or seven of the mutations E421R, D425K, 545K, E149K, N78H, Al 12K, and / or T548R, and at least one other mutation listed in Table A2 in SEQ ID NO: 7. For example, the IscB protein comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, at least five, at least six or seven of the mutations E421R, D425K, 545K, E149K, N78H, Al 12K, and / or T548R, and at least one other mutation listed in Table A2 in SEQ ID NO: 7, and where the at least one other mutation is not E421R, D425K, 545K, E149K, N78H, Al 12K, and / or T548R.

[0024] In some embodiments, the IscB protein one, two, three or four of the mutations E149K, N78H, Al 12K, and / or T548R in SEQ ID NO: 7. For example, the IscB protein one, two, three or four of the mutations E149K, N78H, Al 12K, and / or T548R, and at least one other mutation listed in Table A2 in SEQ ID NO: 7.

[0025] Without wishing to be bound by a theory, in some embodiments, the IscB protein exhibits an increased activity on a target sequence that does not comprise the canonical Target Adjacent Motif (TAM) sequence 5’-ATAAA-3’ at its 3' end as compared to IscB as provided by SEQ ID NO: 2. For example, the IscB protein exhibits an activity that is at least 5 -fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of IscB as provided by SEQ ID NO: 2 on atarget sequence having a 3' end that is not directly adjacent to the canonical TAM sequence (5’-ATAAA-3’). The activity can be measured by a nuclease assay, a deamination assay, or a transcriptional activation assay.

[0026] In some embodiments, the IscB protein described herein exhibits an increased activity on a target sequence comprising, at its 3' end, the TAM sequence RTR (e.g., ATR) as compared to SEQ ID NO: 2, (where R is purine (e.g., A or G) and R is A or C. For example, the IscB protein described herein exhibits an increased activity on a target sequence comprising, at its 3' end, a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM, ATRRM, ATRMA, ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR at its 3 ' end as compared to IscB as provided by SEQ ID NO: 2, wherein R is purine (e.g., A or G) and R is A or C.

[0027] In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H478X, H479N, I564X, T565X, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2, wherein X is any amino acid. In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H478N, H479N, I564K, I564H, I564N, I564Q, I564C, I564M, I564T, I564D, T565R, T565K, T565H, T565C, T565D, T565G, H478C, H478P, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2.

[0028] Without wishing to be bound by a theory, the IscB described herein exhibits an increased activity on a target sequence comprising the TAM sequence ATR. For example, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H478N, H479N, I564K, I564H, I564N, I564Q, I564C, I564M, I564T, I564D, T565R, T565K, T565H, T565C, T565D, T565G, H478C, H478P, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2, and exhibits an increased activity on a target sequence comprising the TAM sequence ATR compared to the activity of SEQ ID NO: 2.

[0029] In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H490X, H49 IN, I576X, T577X, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 8, wherein X is any amino acid. In some embodiments, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H490N, H491N, I576K, I576H, I576N, I576Q, I576C, I576M, I576T, I576D, T577R, T577K, T577H, T577C, T577D, T577G, H490C, H490P, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 8.

[0030] Without wishing to be bound by a theory, the IscB described herein exhibits an increased activity on a target sequence comprising the TAM sequence ATR. For example, the IscB comprises an amino acid sequence comprising at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of H490N, H491N, I576K, I576H, I576N, I576Q, I576C, I576M, I576T, I576D, T577R, T577K, T577H, T577C, T577D, T577G, H490C, H490P, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 8, and exhibits an increased activity on a target sequence comprising the TAM sequence ATR compared to the activity of SEQ ID NO: 8.

[0031] In some embodiments, the IscB protein comprises an amino acid sequence comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least amino sequence swap at position 560-571 in SEQ ID NO: 2. For example, the IscB protein comprises an amino acid sequence comprising at least 2, at least 3, at least 4, at least 5 or at least six amino sequence swap at position Q563-K568 in SEQ ID NO. 2. Without wishing to be bound by a theory, a sequence swap at positions 560-571 (e.g., at position 563-568) leads to enhanced or increased activityon a target sequence comprising the TAM sequence RTAAA. For example, the IscB protein comprises an amino acid sequence comprising the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR or QKDKGA at position Q563-K568 in the SEQ ID NO: 2, and wherein the IscB protein exhibits an increased activity on a target sequence comprising the TAM sequence RTAAA.

[0032] In some embodiments, the IscB protein comprises an amino acid sequence comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least amino sequence swap at position 572-583 in SEQ ID NO: 8. For example, the IscB protein comprises an amino acid sequence comprising at least 2, at least 3, at least 4, at least 5 or at least six amino sequence swap at position Q575-K580 in SEQ ID NO. 8. Without wishing to be bound by a theory, a sequence swap at positions 572-583 (e.g., at position 575-580) leads to enhanced or increased activity on a target sequence comprising the TAM sequence RTAAA. For example, the IscB protein comprises an amino acid sequence comprising the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR or QKDKGA at position Q575-K580 in the SEQ ID NO: 8, and wherein the IscB protein exhibits an increased activity on a target sequence comprising the TAM sequence RTAAA.

[0033] Without wishing to be bound by a theory, the IscB proteins described herein exhibit lower off-target activity as compared to an off-target activity of the IscB domain as provided by SEQ ID NO: 2 or 8.

[0034] In another aspect, provided herein is a fusion protein comprising an IscB protein described herein and an effector domain (also referred to as a functional domain herein). Some exemplary effector domains include, but are not limited to, domains or functional fragments thereof comprising nuclease activity, nickase activity, recombinase activity, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activation activity, or transcriptional repression activity.

[0035] In some embodiments, the effector domain is a nucleic acid editing domain. For example, the effector domain is reverse transcriptase, a deaminase, or a functional fragment thereof. In some embodiments, the effector domain is a reverse transcriptase or a functional fragment thereof.

[0036] In some embodiments, the effector domain is a deaminase domain or a functional fragment thereof. In some embodiments, the deaminase domain is a cytidine deaminase domain or a functional fragment thereof. A preferred cytidine deaminase domain is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase or a functional fragment thereof. In some embodiments, the cytidine deaminase domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the sequences shown in Table DI.

[0037] In some embodiments, the deaminase domain is an adenosine deaminase domain or a functional fragment thereof. In some embodiments, the adenosine deaminase domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the sequences shown in Table D2. In some embodiments, the adenosine deaminase is an ecTadA domain, or variant thereof.

[0038] In some embodiments, the fusion protein comprises two adenosine deaminase domains. The first and second adenosine domains can comprise independently an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the sequences shown in Table D2. In some embodiments, the first adenosine deaminase domain and the second adenosine deaminase domain comprises an ecTadA domain, or variant thereof.

[0039] In some embodiments, the effector domain is a uracil glycosylase inhibitor (UGI) domain or a functional fragment thereof. The UGI domain can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the sequences shown in Table E.

[0040] In another aspect provided herein is complex comprising an IscB protein described herein and a guide nucleic acid (e.g., guide RNA or coRNA). For example, the complex comprises a fusion protein described herein bound / complexed with a guide nucleic acid. Without wishing to be bound by a theory, the complex can be useful for editing a point mutation in a target sequence.

[0041] The guide nucleic acids are described herein below. Generally, the guide nucleic acid is from about 15 nucleotides to about 100 nucleotides in length. For example, the guide nucleic acid can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. in length. Typically, the guide nucleic acid comprises a sequence (guide sequence) of at least 10 contiguous nucleotides that is complementary to a target sequence. For example, the guide nucleic acid comprises a sequence (guide sequence) of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence.

[0042] In some embodiments, the guide nucleic acid is from about 15 nucleotides to about 100 nucleotides in length and comprises a sequence (guide sequence) of at least 10 contiguous nucleotides that is complementary to a target sequence.

[0043] The target sequence can be a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a mammal. In some embodiments, the target sequence is a sequence in the genome of a human.

[0044] In some embodiments, the target sequence comprises a sequence associated with a disease or disorder. For example, the target sequence comprises a point mutation associated with a disease or disorder. The point mutation can be located between about 10 to about 20 nucleotides upstream of the TAM in the target sequence.

[0045] In some embodiments, the target sequence comprises a T to C point mutation. In some embodiments, a complex described herein deaminates the target C point mutation, and wherein the deamination results in a sequence that is not associated with a disease or disorder. In some embodiments, the target C point mutation is present in the DNA strand that is not complementary to the guide RNA. In some embodiments, the target sequence comprises a G to A point mutation. In some embodiments, the complex deaminates the target A point mutation, and wherein the deamination results in a sequence that is not associated with a disease or disorder. In some embodiments, the target A point mutation is present in the DNA strand that is not complementary to the guide RNA.

[0046] In some embodiments, the complex exhibits increased deamination efficiency of a point mutation in a target sequence that does not comprise the canonical TAM (5’-ATAAA-3’) at its 3' end as compared to the deamination efficiency of a complex comprising IscB protein as provided by SEQ ID NO: 2. In some embodiments, the complex exhibits increased deamination efficiency of a point mutation in a target sequence having a 3 ' end that is not directly adj acent to the canonical TAM sequence (5’-ATAAA-3’) that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000- fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the deamination efficiency of complex comprising the IscB protein as provided by SEQ ID NO: 2 on the same target sequence. In some embodiments, a deamination activity is measured using a deamination assay, PCR, or sequencing.

[0047] In some embodiments, the complex produces fewer indels in a target sequence that does not comprise the canonical TAM (5’-ATAAA-3’) at its 3' end as compared to the amount of indels produced by a complex comprising IscB protein as provided by SEQ ID NO: 2 or 8. In some embodiments, the complex produces fewer indels in a target sequence having a 3 ' end that is not directly adjacent to the canonical TAM sequence (5’-ATAAA-3’j that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5 -fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000- fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000- fold, or at least 1,000,000-fold lower as compared to the amount of indels produced by a complex comprising IscB protein as provided by SEQ ID NO: 2 or 8 on the same target sequence. In some embodiments, indels are measured using high-throughput sequencing.

[0048] In some embodiments, the complex exhibits a decreased off-target activity as compared to the off-target activity of a complex comprising IscB protein as provided by SEQ ID NO: 2 or 8. In some embodiments, the off-target activity of the complex is at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold decreased as compared to the off-target activity of a complex comprising IscB protein as provided by SEQ ID NO: 2 or 8. In some embodiments, the target sequence is in the genomeof an organism. In some embodiments, the organism is a prokaryote. In some embodiments, the prokaryote is a bacterium. In some embodiments, the organism is a eukaryote. In some embodiments, the organism is a plant or fungus. In some embodiments, the organism is a vertebrate. In some embodiments, the vertebrate is a mammal. In some embodiments, the mammal is a human. In some embodiments, the organism is a cell. In some embodiments, the cell is a human cell.

[0049] In another aspect described herein is a method comprising contacting a nucleic acid with the fusion protein of any one of the embodiments described herein, and with a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

[0050] In another aspect described herein is a method comprising contacting a cell with the fusion protein of any one of the embodiments described herein, and with a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

[0051] In another aspect described herein is a method comprising contacting a nucleic acid with the complex of any one of the embodiments described herein.

[0052] In another aspect described herein is a method comprising contacting a cell with the complex of any one of the embodiments described herein.

[0053] In some embodiments in any of the aspects, the contacting is performed in vitro. In some other embodiments in any of the aspects, the contacting is performed in vivo.

[0054] In another aspect described herein is a method comprising administering to a subject the fusion protein of any one of the embodiments described herein, and a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

[0055] In another aspect described herein is method comprising administering to a subject the complex of any one of the embodiments described herein.

[0056] In some embodiments, the target sequence of the nucleic acid is a DNA sequence. In some embodiments, the 3' end of the target sequence is not immediately adjacent to the canonical TAM sequence (5’-ATAAA-3’j. In some embodiments, the target sequence comprises a sequence associated with a disease or disorder. In some embodiments, the target DNA sequence comprises a point mutation associated with a disease or disorder.

[0057] In some embodiments, the activity of the fusion protein, or the activity of the complex, results in a correction of the point mutation.

[0058] In some embodiments, the target DNA sequence comprises a T to C point mutation associated with a disease or disorder, and wherein the deamination of the mutant C base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence encodes a protein and wherein the point mutation is in a codon and results in a change in theamino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant C results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant C results in the codon encoding the wild-type amino acid.

[0059] In some embodiments, the target DNA sequence comprises a G to A point mutation associated with a disease or disorder, and wherein the deamination of the mutant A base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence encodes a protein and wherein the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant A results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant A results in the codon encoding the wild-type amino acid.

[0060] In some embodiments, the contacting is in vivo in a subject. In some embodiments, the subject has or has been diagnosed with a disease or disorder. In some embodiments, the disease or disorder is a proliferative disease, a genetic disease, a neoplastic disease, a metabolic disease, or a lysosomal storage disease.

[0061] In another aspect described herein is a kit comprising a nucleic acid construct, comprising: (a) a nucleic acid sequence encoding the fusion protein of any one of the embodiments described herein; and (b) a heterologous promoter that drives expression of the sequence of (a).

[0062] In another aspect described herein is a kit comprising a nucleic acid construct, comprising:(a) a nucleic acid sequence encoding the complex of any one of the embodiments described herein; and(b) a heterologous promoter that drives expression of the sequence of (a).

[0063] In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.

[0064] In another aspect described herein is a polynucleotide encoding the fusion protein of any one of the embodiments described herein or the complex of any one of the embodiments described herein.

[0065] In another aspect described herein is a vector comprising a polynucleotide of any of the embodiments described herein.

[0066] In some embodiments, the vector comprises a heterologous promoter driving expression of the polynucleotide encoding the fusion protein or the polynucleotide encoding the complex.

[0067] In another aspect described herein is a method comprising contacting a cell with the vector of any of the embodiments described herein.

[0068] In some embodiments, the vector is transfected into the cell. In some embodiments, the vector is transfected into the cell using electroporation, heat shock, or a composition comprising a cationic lipid.

[0069] In another aspect described herein is a cell comprising the fusion protein of any one of the embodiments described herein, or a nucleic acid molecule encoding the fusion protein of any one of the embodiments described herein.

[0070] In another aspect described herein is a cell comprising the complex of any one of the embodiments described herein, or a nucleic acid molecule encoding the complex of any one of the embodiments described herein.

[0071] In another aspect described herein is a cell comprising the vector of any one of the embodiments described herein.

[0072] In some embodiments of any one of the aspects described herein, an IscB protein described herein has a non-canonical TAM specificity. Stated in another way, an IscB protein described herein can act on a target sequence that does not have the canonical TAM sequence 5’-ATAAA-3’ at its 3' end.

[0073] In another aspect, provided herein is a method of modifying a target nucleic acid, the method comprising contacting an IscB (or a fusion protein) described herein and a guide nucleic acid with a target sequence.

[0074] In some embodiments, the target nucleic acid is in a cell and the method comprises administering to the cell a DNA and RNA targeting system or composition of any one of the preceding embodiments. In some embodiments, said modifying the target nucleic acid comprises cleaving the target nucleic acid. In some embodiments, said cleaving the target nucleic acid forms at least one 5’- overhang. In some embodiments, said modifying the target nucleic acid comprises deletion, insertion or change of a nucleotide in the target nucleic acid. In some embodiments, said modifying the target nucleic acid comprises editing (e.g., changing) one or more nucleobases; correcting or introducing a premature stop codon, disrupting a splice site; inserting or restoring a splice site, inserting a gene or gene fragment into the target nucleic acid, or a combination thereof.

[0075] In some embodiments of any of the aspects, the target nucleic acid is DNA. In some preferred embodiments, the target nucleic acid is genomic DNA, episomal DNA, viral DNA, or bacterial DNA. In some embodiments of any of the aspects, the target nucleic acid is double-stranded DNA. In some other embodiments of any of the aspects, the target nucleic acid is single-stranded DNA.

[0076] In some embodiments of any of the aspects, the target nucleic acid is RNA. In some other embodiments of any of the aspects, the target nucleic acid is double -stranded RNA. In some other embodiments, the target nucleic acid is single-stranded RNA. In some embodiments, the target nucleic acid is RNA selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small nuclear RNA (snRNA), smallnucleolar RNA (snoRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmatic RNA (scRNA).

[0077] In some embodiments of any of the aspects described herein, the target nucleic acid is a disease- associated nucleic acid sequence.

[0078] In another aspect, provided herein is an isolated nucleic acid modified by a method of anyone of the preceding embodiments.

[0079] In another aspect, provided herein is an isolated cell or progeny thereof comprising a nucleic acid modified by a method of anyone of the preceding embodiments

[0080] Generally, the IscB proteins and the guide nucleic acid described herein do not naturally occur together.

[0081] Described herein are DNA and RNA targeting enzymes and systems, that are useful for modifying target nucleic acid sequences. Accordingly in one aspect is a DNA and RNA targeting composition comprising an IscB protein of describe herein and a guide nucleic acid capable of binding with the IscB protein, wherein the guide nucleic acid comprises a guide sequence complementary to target sequence and capable of directing the IscB protein to a target polynucleotide.

[0082] In some embodiments in any of the aspects, the guide nucleic acid is from about 10 nucleotides to about 150 nucleotides in length, preferably 12 to 50 nt, more preferably 15 and 45 nt in length. In some embodiments, the guide nucleic acid further comprises an aptamer. In some embodiments in any of the aspects described herein, the guide nucleic acid further comprises an extension to add an RNA template.

[0083] In some embodiments of any of the aspects described herein, the target sequence comprises a target adjacent motif (TAM) sequence 3’ of the target sequence. In some embodiments of any of the aspects, the target polynucleotide is DNA. In some embodiments, the target sequence comprises a TAM sequence of ATR, where R is purine (e.g., G or A).

[0084] In some embodiments, the composition further comprises a functional domain associated with the IscB protein.

[0085] In some embodiments, the functional domain has transposase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double- strand DNA cleavage activity, nucleic acid binding activity, detectable activity, or any combination thereof. In some embodiments, the functional domain is covalently linked with the IscB protein, wherein the IscB is a fusion protein described herein.

[0086] In some embodiments, the composition further comprises a serine or tyrosine recombinase.

[0087] In some embodiments, the composition further comprises a homologous recombination donor template comprising a donor sequence for insertion into a target polynucleotide.

[0088] In some embodiments, a nuclease domain of the IscB protein is catalytically inactive. In some other embodiments, the nuclease domain has nickase activity or is engineered to have nickase activity.

[0089] In another aspect described herein is an engineered cell comprising the composition of any of the embodiments described herein.

[0090] In another aspect described herein is a method of modifying a target sequence in a cell, the method comprising administering a composition of any of the embodiments described herein to the cell.

[0091] In another aspect described herein is a method of modifying a target sequence in a cell, the method comprising administering to the cell a composition comprising an IscB protein and / or a guide nucleic acid of any of the embodiments described herein, wherein IscB protein and / or the guide nucleic acid are provided via one or more polynucleotides encoding the IscB and / or guide nucleic acid, and wherein the one or more polynucleotides are operably configured to express the IscB / or the guide nucleic acid. In some embodiments, the modifying comprises cleaving a DNA polynucleotide. In some preferred embodiments, the cleaving results in 5’ overhangs.

[0092] In another aspect described herein is a method of targeting a polynucleotide, comprising contacting a sample that comprises a target polynucleotide with the composition of any of the embodiments described herein, or one or more polynucleotides encoding one or more components of the composition of any of the embodiments described herein. In some embodiments, contacting results in modification of a gene product or modification of the amount or expression of a gene product. In some preferred embodiments, the target sequence of the polynucleotide is a disease-associated target sequence.

[0093] In another aspect described herein is a composition comprising: the IscB protein of any one of claims 1-32, wherein the IscB protein is catalytically inactive, a nucleotide deaminase associated with or otherwise capable of forming a complex with the IscB protein, and a guide nucleic acid capable of forming a complex with the IscB protein and directing site -specific binding at a target sequence. In some embodiments, the nucleotide deaminase is an adenosine deaminase or a cytidine deaminase.

[0094] In another aspect described herein is a cell or progeny thereof genetically engineered to express one or more components of the composition of any one of the embodiments described herein.

[0095] In another aspect described herein is a method of editing nucleic acids in target polynucleotides comprising delivering the composition of any of the embodiments described herein, or one or more polynucleotides encoding one or more components of the composition, to a cell or population of cells comprising the target sequence. In some embodiments, the target polynucleotides are target sequences within genomic DNA. In some preferred embodiments, the target polynucleotide is edited at one or more bases to introduce a G->A or C->T mutation.

[0096] In another aspect is an isolated cell or progeny thereof comprising one or more base edits made using the method of any one of the embodiments described herein.

[0097] In another aspect as described herein is a composition comprising: the IscB protein of any one of the embodiments described herein, wherein the IscB is catalytically inactive, a reverse transcriptase associated with or otherwise capable of forming a complex with the IscB protein, and a guide nucleic acid capable of forming a complex with the IscB protein and directing site-specific binding of the IscB to a target sequence, and wherein the guide nucleic further comprises a donor sequence for insertion into the target sequence.

[0098] In another aspect as described herein is a method of modifying target polynucleotides comprising delivering the composition of any of the embodiments described herein, or one or more polynucleotides encoding one or more components of the composition to a cell, wherein the complex directs the reverse transcriptase to the target sequence and the reverse transcriptase facilitates insertion of the donor sequence from the guide nucleic acid into the target sequence. In some embodiments of any of the aspects, insertion of the donor sequence: introduces one or more base edits; corrects or introduces a premature stop codon; disrupts a splice site; inserts or restores a splice site; inserts a gene or gene fragment at one or both alleles of the target polynucleotide; or a combination thereof.

[0099] In another aspect as described herein is an isolated cell or progeny thereof comprising the modifications made using the method of any of the embodiments described herein.

[0100] In another aspect as described herein is a composition comprising: the IscB protein of any one of the embodiments described herein; a non-LTR retrotransposon protein associated with or otherwise capable of forming a complex with the IscB protein; guide nucleic acid capable of forming a complex with the IscB protein and directing site-specific binding to a target sequence; and a donor construct comprising a donor polynucleotide for insertion to the target sequence and located between two binding elements capable of forming a complex with the non-LTR retrotransposon protein.

[0101] In some embodiments, the IscB protein is fused to the N-terminus of the non-LTR retrotransposon protein. In some embodiments, the IscB protein is engineered to have nickase activity. In some embodiments, the guide nucleic acid directs the fusion protein to a target sequence 5 ’ of the targeted insertion site, and wherein the IscB protein generates a double-strand break at the targeted insertion site. In some other embodiments, the guide nucleic acid directs the fusion protein to a target sequence 3 ’ of the targeted insertion site, and wherein the IscB protein generates a double-strand break at the targeted insertion site.

[0102] In some embodiments, the donor polynucleotide further comprises a polymerase processing element to facilitate 3’ end processing of the donor polynucleotide sequence. In some other embodiments, the donor polynucleotide further comprises a homology region to the target sequence on the 5’ end of the donor construct, the 3’ end of the donor construct, or both. In some embodiments of any of the aspects, the homology region is from 8 to 25 base pairs.

[0103] In another aspect as described herein is a method of modifying target polynucleotides comprising delivering the composition of any one of the embodiments described herein, or one or more polynucleotides encoding one or more components of the composition to a cell, wherein the complex directs the non-LTR retrotransposon protein to the target sequence and the non-LTR retrotransposon protein facilitates insertion of the donor polynucleotide sequence from the donor construct into the target polynucleotide.

[0104] In another aspect as described herein is an isolated cell or progeny thereof comprising the modifications made using the method of any of the embodiments described herein.

[0105] In one aspect, provided herein is a guide RNA (also referred to as a coRNA) comprising a conserved portion of a coRNA (parent coRNA) comprising one or more stem-loop structures, wherein at least one of the stem-loop structures comprises a modification, and wherein a conserved portion of the coRNA is shorter than the conserved portion of the parent coRNA and is capable of binding with a transposon family encoded nuclease or a homolog or ortholog thereof.

[0106] It is noted that coRNA is also referred to as omegaRNA herein and the terms coRNA and omegaRNA are used interchangeably herein.

[0107] In some embodiments, the conserved region of the coRNA comprises at least two stem -loop structures and wherein said modification is in a stem-loop structure near to the 5 ’-end (5 ’-stem -loop) of the conserved region coRNA.

[0108] In some embodiments of any one of the aspects described herein, said modification comprises deletion of from about 3 to about 46 nucleotides from the 5 ’-stem-loop. In some embodiments of any one of the aspects described herein, said modification comprises deletion of 3, 7, 11, 15, 18, 22, 26, 30, 34, 37 or 41 nucleotides from the 5’-stem-loop. In some preferred embodiments of any one of the aspects described herein, said modification comprises deletion of 15, 18, 30, 37 or 41 nucleotides from the 5 ’-stem-loop. In some embodiments of any one of the aspects described herein, said modification comprises deletion of 8, 12, 16, 20, 23, 27, 31, 35, 39, 42 or 46 nucleotides from the 5 ’-stem-loop. In some preferred embodiments of any one of the aspects described herein, said modification comprises deletion of 20, 23, 35, 42 or 46 nucleotides from the 5 ’-stem-loop.

[0109] In some embodiments, nucleotides at position 12-57, 14-55, 16-54, 18-52, 20-50, 22-48, 24-46, 26-45, 28-43, 30-41 or 32-39 are deleted (counting from 5’-end, e.g., 5’-end of the conserved region of the parent coRNA). In some embodiments, nucleotides at position 12-57, 14-55, 18-52, 24-46, or 26-45 are deleted (counting from 5’-end, e.g., 5’-end of the conserved region of the parent coRNA). In some embodiments, the deleted nucleotides are replaced with a linker (e.g., a loop portion). In some embodiments, the linker is single-stranded nucleic acid comprising from about from about 4 nucleotides to about 15 nucleotides. In some embodiments, a loop portion of the 5’-stem-loop comprises from 4,5 or 6 nucleotides. In some embodiments, a loop portion of the 5 ’-stem-loop comprises the nucleotide sequence GAAA or GAAAA.

[0110] In some embodiments, at least a portion of the 5 ’-stem -loop is within 45 nucleotides or within 25 nucleotides, counting from 5 ’-end, e.g., 5 ’-end of the conserved region of the parent coRNA. In some embodiments, a stem portion of the 5 ’-stem-loop comprises a double-stranded region comprising from about 8 nucleotide base-pairs about 9 nucleotide base-pairs, about 10 nucleotide base- pairs, about 11 nucleotide base-pairs, about 12 nucleotide base-pairs, about 13 nucleotide base-pairs, about 14 nucleotide base-pairs, about 15 base pairs nucleotide base-pairs, about 16 base pairs nucleotide base-pairs, about 17 base pairs nucleotide base-pairs, about 18 base pairs nucleotide base-pairs, about 19 base pairs nucleotide base-pairs, about 20 base pairs nucleotide base-pairs, or about 25 base pairs nucleotide base-pairs. In some preferred embodiments, a stem portion of the 5 ’-stem-loop comprises a double -stranded region comprises 10 nucleotide base-pairs.

[0111] In some embodiments, the 5’-stem-loop comprises from about 20 nucleotides, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, or about 65 nucleotides. In some preferred embodiments, the 5 ’-stem-loop comprises 23, 24, 25, 26 or 27 nucleotides. In some embodiments of any of the aspects, the 5 ’-stem-loop comprises a nucleic acid modification, optionally selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof.

[0112] In some embodiments, a stem (double-stranded) region of the 5 ’-stem-loop comprises a nucleic acid modification. In some other embodiments, a loop (single-stranded) region of the 5 ’-stem- loop comprises a nucleic acid modification.

[0113] In some embodiments, said nucleic acid modification is a modified nucleotide selected from the group consisting of 2’-O-methyl (2’-OMe) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’-C-methylene (locked nucleic acid, LNA) nucleotides, 2’- O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl -carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene (Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-M0E) nucleotides, 2’-O-allyl nucleotides, 2’-C-allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-O- NMA) nucleotides, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, 2’-F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3-dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; a non-natural or modified nucleobase; or a combination thereof.

[0114] In some embodiments, a stem (double-stranded) region of the 5 ’-stem -loop comprises a 2’- O-methyl modified nucleotide. In some other embodiments, a stem (double-stranded) region of the 5’- stem-loop comprises a 2’-fluoro modified nucleotide. In some embodiments, a nucleotide in a stem (double-stranded) region of the 5 ’-stem -loop comprises a non-natural or modified nucleobase.

[0115] In some embodiments, a stem (double-stranded) region of the 5 ’-stem-loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkage (e.g., a modified intemucleoside linkage selected independently from the group consisting of phosphorothioates (R. S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino ( — CH2- N(CHs)-0 — CH2-), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2-0 — and dialkylsiloxane), N,N'-dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’), C3’-CH2-NH-NH-C5’, 3'- NHP(O)(OCH3)-O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ intemucleoside linkages, 2’->3’ intemucleoside linkages, 3 ’->3’ intemucleoside linkages, and 5 ’->5’ intemucleoside linkages, optionally the modified intemucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified intemucleoside linkage is phosphorothioate (PS).

[0116] In some embodiments, a loop (single-stranded) region of the 5 ’-stem-loop comprises a 2’- O-methyl modified nucleotide. In some other embodiments, a loop (single-stranded) region of the 5’- stem-loop comprises a 2’-fluoro modified nucleotide.

[0117] In some embodiments, a nucleotide in a loop (single -stranded) region of the 5 ’-stem-loop comprises a non-natural or modified nucleobase.

[0118] In some embodiments, a loop (single-stranded) region of the 5 ’-stem -loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkage (e.g., a modified intemucleoside linkage selected independently from the group consisting of phosphorothioates (R. S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino ( — CH2- N(CH3)-0 — CH2-), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2-0 — and dialkylsiloxane), N,N'-dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'),formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’), C3’-CH2-NH-NH-C5’, 3'- NHP(O)(OCH3)-O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ intemucleoside linkages, 2’->3’ intemucleoside linkages, 3 ’->3’ intemucleoside linkages, and 5 ’->5’ intemucleoside linkages, optionally the modified intemucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified intemucleoside linkage is phosphorothioate (PS).

[0119] In some embodiments, a stem (double-stranded) region of the 5 ’-stem-loop comprises a duplex stabilizing modification, optionally the duplex stabilizing modification is 2’-F nucleotide, 2’- OMe nucleotide, 2 ’-methoxy ethyl nucleotide, 2,6-diaminopurine nucleotide, 5-methyl cytidine, N4- ethyl cytidine, 5-propynyl cytidine, 5-propynyl uridine, 5 -hydroxybutynl-2’ -deoxyuridine, 8-aza-7- deazaguanosine, a locked nucleic acid (LNA), and / or covalent cross-linking of two strands of the duplex.

[0120] In some embodiments, two nucleotides in the stem of the 5 ’-loop-stem are covalently linked to each other via a position other than 5 ’and 3 ’ .

[0121] In some embodiments, the conserved region of the coRNA comprises at least two stem -loop stmctures and wherein said modification is in a stem-loop structure proximal to the 3 ’-end (3 ’-stem- loop) of the conserved region of the coRNA.

[0122] In some embodiments, said modification comprises deletion of from about 1 to about 22 nucleotides from the 3’-stem-loop. In some preferred embodiments, said modification comprises deletion of 2, 6, 8, 10, 12, 16, or 20 nucleotides from the 3’-stem-loop. In some embodiments, said modification comprises deletion of 2, 10, 12, or 16 nucleotides from the 3 ’-stem-loop. In some embodiments, said modification comprises deletion of 2, 10, 12 or 16 nucleotides from the 5’-stem- loop. In some embodiments, said modification comprises deletion of 2, 10, 12, or 16 nucleotides from the 3 ’-stem -loop.

[0123] In some embodiments, nucleotides at position 159-172, 160-171, 161-170, 163-168, 158- 173, 156-175 or 154-177 are deleted (counting from 5’-end, e.g., of the parent coRNA). In some other embodiments, nucleotides at position 159-172, 163-168, 158-173, or 156-175 are deleted (counting from 5’-end, e.g., of the parent coRNA).

[0124] In some embodiments, the deleted nucleotides in the 3 ’-stem -loop are replaced with a linker (e.g., a loop portion). In some embodiments, the linker is a single-stranded nucleic acid comprising from about from about 4 nucleotides to about 15 nucleotides.

[0125] In some embodiments, a loop portion of the 5 ’-stem-loop comprises from 4, 5 or 6 nucleotides. In some embodiments, a loop portion of the 5 ’-stem-loop comprises the nucleotide sequence GAAA or GAAAA.

[0126] In some embodiments, at least a portion of the 3 ’-stem-loop is within 50 nucleotides, counting from 3 ’-end of the modified coRNA. In some other embodiments, at least a portion of the stem of the 5 ’-stem- loop is within 45 nucleotides, counting from 3 ’-end of the modified coRNA.

[0127] In some embodiments, a stem portion of the 3 ’-stem-loop comprises a double -stranded region comprising from about 3 nucleotide base-pairs to about 10 nucleotide base-pairs. In some other embodiments, a stem portion of the 3 ’-stem-loop comprises a double-stranded region comprising about 3 nucleotide base-pairs, about 4 nucleotide base-pairs, about 5 nucleotide base-pairs, about 6 nucleotide base-pairs, about 7 nucleotide base-pairs, or about 8 nucleotide base-pairs. In some preferred embodiments, a stem portion of the 3 ’-stem-loop comprises a double-stranded region comprising about 3 nucleotide base-pairs, about 4 nucleotide base-pairs, about 5 nucleotide base-pairs, or about 6 nucleotide base -pairs.

[0128] In some embodiments, a stem portion of the 5 ’-stem-loop comprises a double -stranded region comprises 3 or 4 nucleotide base-pairs.

[0129] In some embodiments, the 3 ’-stem-loop comprises from about 8 nucleotides, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides. In some preferred embodiments, the 3’-stem-loop comprises 9, 10, 11, 12, 13, 14 or 15 nucleotides. In some embodiments of any of the aspects described herein, the 3 ’-stem-loop comprises a nucleic acid modification, optionally selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof.

[0130] In some embodiments, a stem (double-stranded) region of the 3 ’-stem-loop comprises a nucleic acid modification. In some embodiments, a loop (single -stranded) region of the 3 ’-stem-loop comprises a nucleic acid modification. In some embodiments of any of the aspects described herein, said nucleic acid modification is a modified nucleotide selected from the group consisting of 2’-O- methyl (2’-OMe) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’-C-methylene (locked nucleic acid, LNA) nucleotides, 2’-O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl-carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene (Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-M0E) nucleotides, 2’-O-allyl nucleotides, 2’-C- allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-0-NMA) nucleotides, a 2'-O- dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, 2’- F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; a non-natural or modified nucleobase; or a combination thereof.

[0131] In some embodiments, a stem (double-stranded) region of the 3’-stem-loop comprises a 2’- O-methyl modified nucleotide. In some other embodiments, a stem (double-stranded) region of the 3’- stem-loop comprises a 2’-fluoro modified nucleotide. In some embodiments, a nucleotide in a stem (double-stranded) region of the 5’-stem-loop comprises a non-natural or modified nucleobase. In some embodiments, a stem (double-stranded) region of the 3’-stem-loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified internucleoside linkage (e.g., a modified internucleoside linkage selected independently from the group consisting of phosphorothioates (R, S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)2- O— and dialkylsiloxane), N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2- C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2- O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’), C3’-CH2-NH-NH-C5’, 3'- NHP(O)(OCH3)-O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ internucleoside linkages, 2’->3’ internucleoside linkages, 3’->3’ internucleoside linkages, and 5’->5’ internucleoside linkages, optionally the modified internucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified internucleoside linkage is phosphorothioate (PS).

[0132] In some embodiments, a loop (single-stranded) region of the 5’-stem-loop comprises a 2’- O-methyl modified nucleotide. In some other embodiments, a loop (single-stranded) region of the 5’- stem-loop comprises a 2’-fluoro modified nucleotide. In some embodiments, a nucleotide in a loop (single-stranded) region of the 3’-stem-loop comprises a non-natural or modified nucleobase. In some embodiments, a loop (single-stranded) region of the 5’-stem-loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified internucleoside linkage (e.g., a modified internucleoside linkage selected independently from the group consisting of phosphorothioates (R, S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)2- O— and dialkylsiloxane), N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2- C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2- O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’),thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’), C3’-CH2-NH-NH-C5’, 3'- NHP(O)(OCH3)-O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ intemucleoside linkages, 2’->3’ intemucleoside linkages, 3 ’->3’ intemucleoside linkages, and 5 ’->5’ intemucleoside linkages, optionally the modified intemucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified intemucleoside linkage is phosphorothioate (PS). In some embodiments, two nucleotides in the stem of the 5 ’-loop-stem are covalently linked to each other via a position other than 5 ’and 3’.

[0133] In some embodiments, the conserved region of the coRNA comprises at least three stem- loop stmctures. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) nucleotide in a loop region of a stem-loop forms a base-pair with a nucleotide outside said stem-loop. At least one (e.g., one or both) member of the base-pair comprises a modification capable of enhancing base-pairing. In some embodiments, the base-pair nucleotide in the loop comprises the modification capable of enhancing base-pairing. In some other embodiments, the base-pair nucleotide outside said stem -loop comprises the modification capable of enhancing base-pairing.

[0134] In some embodiments, said modification capable of enhancing base-pairing is 2’-F modified nucleotide, 2’-OMe modified nucleotide, 2 ’-methoxy ethyl modified nucleotide, 2,6- diaminopurine nucleotide, 5-methyl cytidine, N4-ethyl cytidine, 5-propynyl cytidine, 5-propynyl uridine, 5 -hydroxybutynl-2’ -deoxyuridine, 8-aza-7-deazaguanosine, or a locked nucleic acid (LNA). In some embodiments, said modification capable of enhancing base-pairing is in at an internal (i.e., not a 5 ’-stem-loop or 3 ’-stem-loop) stem-loop of the coRNA. In some preferred embodiments, said modification capable of enhancing base-pairing is in a loop (single-stranded) region of an internal (i.e., not a 5 ’-stem-loop or 3 ’-stem-loop) stem-loop of the coRNA.

[0135] In some embodiments, the coRNA (e.g., a conserved region) is from about 50 to about 90 nucleotides shorter than the parent coRNA. In some embodiments, the coRNA (e.g., a conserved region) is from about 55 to about 85 nucleotides shorter than the parent coRNA. In some embodiments, the coRNA (e.g., a conserved region) is about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, or about 80 nucleotides shorter than the parent coRNA. In some embodiments, the coRNA (e.g., a conserved region) is from about 130 nucleotides to about 190 nucleotides in length. In some embodiments, the coRNA (e.g., a conserved region) is from about 135 nucleotides to about 180 nucleotides in length. In some embodiments, the coRNA (e.g., a conserved region) is from about 140 nucleotides to about 170 nucleotides in length. In some embodiments, the coRNA (e.g., a conserved region) is from about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 1454, about 155, about 156, about 157, about 158, about 159, or about 160 nucleotides in length.

[0136] In some embodiments, the coRNA comprises a 5’ end modification. In some other embodiments, the coRNA comprises a 3’ end modification. In some embodiments, the coRNA comprisesa 5’ end modification and a 3’ end modification. In some preferred embodiments, the 5’ and / or 3’ end modification comprises a protective end modification where the protective end modification is a modified nucleotide selected from 2’-O-methyl (2’-0Me) modified nucleotide, 2’-O-(2-methoxyethyl) (2’- O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, a 3 ’->3’ linkage between nucleotides, an inverted abasic modified nucleotide, or combinations thereof.

[0137] In some embodiments, the 3’ end modification comprises any one of: a modification of any one or more of the last 7, 6, 5, 4, 3, 2, or 1 nucleotides; one modified nucleotide; two modified nucleotides; three modified nucleotides; four modified nucleotides; five modified nucleotides; six modified nucleotides; or seven modified nucleotides. In some other embodiments, the 3’ end modification comprises one or more of: a modified intemucleoside linkage (e.g., PS, imidp or MMI linkage) between nucleotides; a 2’-OMe nucleotide; a 2’-0-M0E nucleotide; a 2’ -F nucleotide; a LNA nucleotide; a 3 ’->3’ linkage between nucleotides; an inverted abasic nucleotide; and a combination of one or more of (i.) - (vii.) . In some preferred embodiments, the 3’ end modification comprises one or more of: a modified intemucleoside linkage (e.g., PS, imidp or MMI linkage) between the last and second to last nucleotides; a modified intemucleoside linkage (e.g., PS, imidp or MMI linkage) between the last and second to last nucleotide and a PS linkage between the second and third to last nucleotides; modified intemucleoside linkages (e.g., PS and / or MMI linkage) between any one or more of the last four nucleotides; modified intemucleoside linkages (e.g., PS and / or MMI linkage) between any one or more of the last five nucleotides; or modified intemucleoside linkages (e.g., PS and / or MMI linkage) between any one or more of the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0138] In some embodiments, the 3’ end modification comprises at least one 2’-F nucleotide, 2’- OMe nucleotide, 2 ’-O-MOE nucleotide, inverted abasic nucleotide or a 3 ’->3’ intemucleoside linkage. In some embodiments, the 3’ end modification comprises: a modification of one or more of the last 1- 7 nucleotides, wherein the modification is a modified intemucleoside linkages (e.g., PS and / or MMI linkage), inverted abasic nucleotide, a 3 ’->3’ intemucleoside linkage, 2’-OMe, 2 ’-O-MOE, 2’-F, LNA, or a combination thereof; a modification to the last nucleotide with 2’-OMe, 2’-O-MOE, 2’-F, LNA, or combinations thereof, and an optional one or two modified intemucleoside linkages (e.g., PS and / or MMI linkage) to the next nucleotide; a modification to the last and / or second to last nucleotide with 2’- OMe, 2 ’-O-MOE, 2’-F, LNA, or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage); iv. a modification to the last, second to last, and / or third to last nucleotides with 2’-OMe, 2’-O-moe, 2’-F, or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage); a modification to the last, second to last, third to last, and / or fourth to last nucleotides with 2’-OMe, 2 ’-O-MOE, 2’-F, LNA or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage); or a modification to the last, second to last, third to last, fourth to last, and / or fifth to lastnucleotides with 2’-0Me, 2’-0-M0E, 2’-F, LNA, or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage).

[0139] In some embodiments, the 3’ end modification comprises any one or more of: a 2’-OMe modified nucleotide at the last position, three consecutive 2’-O-MOE modified nucleotides immediately 5’ to the 2’-OMe modified nucleotide, and three consecutive PS linkages between the last three nucleotides; five consecutive 2’-OMe modified nucleotides from the 3 ’ end of the 3 ’ terminus, and three PS linkages between the last three nucleotides; an inverted abasic modified nucleotide at the last position; an inverted abasic modified nucleotide at the last position, and three consecutive 2’-OMe modified nucleotides at the last three positions; 15 consecutive 2’-OMe modified nucleotides from the 3’ end, five consecutive 2’-F modified nucleotides immediately 5’ to the 2’-OMe modified nucleotides, and three PS linkages between the last three nucleotides; alternating 2’-OMe modified nucleotides and 2’-F modified nucleotides at the last 20 nucleotides, and three PS linkages between the last three nucleotides; two or three consecutive 2’-OMe modified nucleotides, and three PS linkages between the last three nucleotides; one PS linkage between the last and next to last nucleotides; and 15 or 20 consecutive 2’-OMe modified nucleotides, and three PS linkages between the last three nucleotides.

[0140] In some embodiments, the 5 ’ end modification comprises any one or more of: one modified nucleotide; two modified nucleotides; three modified nucleotides; four modified nucleotides; five modified nucleotides; six modified nucleotides; and seven modified nucleotides. In some embodiments, the 5 ’ end modification comprises any one or more of: a modification of between 1 and 7, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2 nucleotides. In some embodiments, the 5’ end modification comprises any one or more of: (i) a modified intemucleoside linkage (e.g., a phosphorothioate and / or MMI linkage) between nucleotides; (ii) a 2’-OMe nucleotide; (iii) a 2’-0-M0E nucleotide; (iv) a 2’ -F nucleotide; (v) a LNA; (vi) a 3 ’->3’ linkage; (vii) an inverted abasic modified nucleotide; (viii) a deoxyribonucleotide; (ix) an inosine; and (x) combinations of one or more of (i) - (ix).

[0141] In some other embodiments, the 5’ end modification comprises: 1, 2, 3, 4, 5, 6, and / or 7 modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between nucleotides; or about 1-2, 1-3, 1-4, 1-5, 1-6, or 1-7 modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between nucleotides.

[0142] In some preferred embodiments, the 5’ end modification comprises: there is one modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkage, and the linkage is between nucleotides 1 and 2; there are two modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages, and the linkages are between nucleotides 1 and 2, and 2 and 3; there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, and 3 and 4; there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, and 4 and 5; there are modified intemucleoside(e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6; there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, and 6 and 7; or there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, and 7 and 8. In some embodiments, the 5’ end modification comprises at least one 2’-0Me, 2’-0-M0E, inverted abasic, or 2’-F modified nucleotide.

[0143] In some embodiments, the coRNA further comprises a guide region capable of hybridizing with a target nucleic acid. In some embodiments, the coRNA is hybridized with a second nucleic acid strand. In some embodiments, the second strand comprises a guide region comprising a sequence capable of hybridizing with a target nucleic acid. In some embodiments, the guide region is from about 15 to about 30 nucleotides in length. In some embodiments, the guide region is from about 18 to about 25 nucleotides in length. In some embodiments, the guide region is from 18, 19, 20, 21, 22 or 23 nucleotides in length. In some embodiments, the guide region is at 5 ’-end of the coRNA. In some embodiments, the guide region is at 3 ’-end of the coRNA.

[0144] In some embodiments, the guide region comprises a nucleic acid modification, optionally selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof.

[0145] In some embodiments, the guide region comprises: a modification of one or more of nucleotides 1-7, wherein the modification is a modified intemucleoside (e.g., phosphorothioate and / or MMI) linkage, 3’->3’ intemucleoside linkage, inverted abasic nucleotide, 2’-OMe, 2’-0-M0E, 2’-F, 2’-H (a deoxyribonucleotide), an inosine, and / or combinations thereof; a modification to the first nucleotide of the guide region with 2’-OMe, 2’-0-M0E, 2’-F, 2’-H, an inosine, or combinations thereof, and an optional modified intemucleoside (e.g., phosphorothioate and / or MMI) linkage to the next nucleotide; a modification to the first and / or second nucleotide of the guide region with 2’-OMe, 2’-0-M0E, 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages; a modification to the first, second, and / or third nucleotides of the guide region with 2’-OMe, 2’-0-M0E 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages; a modification to the first, second, third, and / or fourth nucleotides of the guide region with 2’-OMe, 2’-0-M0E, 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages; or a modification to the first, second, third, fourth, and / or fifth nucleotides of the guide region with 2’-OMe, 2’-0-M0E, 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages.

[0146] In some embodiments, the coRNA comprises any one or more of: 2’-OMe nucleotides at nucleotides 1, 2, and 3 of the guide region; 2’-OMe nucleotides at nucleotides 1, 2, and 3 of the guide region and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; 2’-0Me nucleotides at nucleotides 1, 2, 3, 4, and 5 of the guide region; 2’-0Me nucleotides at nucleotides 1, 2, 3, 4, and 5 of the guide region and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the guide region; 2’-0-M0E modified nucleotides at nucleotides 1, 2, and 3 of the guide region; 2’-0-M0E modified nucleotides at nucleotides 1, 2, and 3 of the guide region and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; an inverted abasic nucleotide at nucleotide 1 of the guide region; an inverted abasic nucleotide at nucleotide 1 of the guide region and 2’-0Me nucleotides at nucleotides 1, 2, and 3 of the guide region; and an inverted abasic nucleotide at nucleotide 1 of the guide region, 2’-0Me nucleotides at nucleotides 1, 2, and 3 of the guide region, and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the variable region.

[0147] In some embodiments, the coRNA comprises one of more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more including all) modified pyrimidine-A dinucleotide (modified YA site). In some embodiments, the coRNA comprises at least one modified YA site in the guide region. In some embodiments, the coRNA comprises at least one modified YA site in a conserved region of the coRNA. In some embodiments, the modified YA site comprises a 2 ’-fluoro nucleotide, optionally the pyrimidine of the YA site; a 2’-H nucleotide, optionally the pyrimidine of the YA site; a 2’-OMe nucleotide, optionally the pyrimidine of the YA site; a PS linkage between the nucleotides; or a combination thereof. In some embodiments, the coRNA comprises: a modified YA site at 1, 2, 3, 4, or 5 guide region YA sites; a modified YA site at 1, 2, 3, 4, or 5 guide region YA sites, wherein the modification of at least one guide region YA site is different from any 5’ end modification of the coRNA; a modified YA site at one or more guide region YA sites that are at or after nucleotide 8 from the 5 ’ end of the 5 ’ terminus; a modified YA site at one or more guide region YA sites that are within nucleotides 5-end, 6-end, 7-end, 8-end, 9- end, or 10-end from the 5’ end of the 5’ terminus; a modified YA site at one or more guide region YA sites that are within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3’ terminal nucleotide of the guide region; a modified YA site at a guide region YA site other than a 5’ end modification; or a modified YA site at a guide region YA site, wherein the modification of the guide region YA site comprises a modification at least one nucleotide located 5’ of the guide region YA site does not comprise.

[0148] In some embodiments, the coRNA comprises: a modified YA site at two or more guide region YA sites that are at or after nucleotide 8 from the 5 ’-end; a modified YA site at two or more guide region YA sites that are within nucleotides 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the5’ end; a modified YA site at two or more guide region YA sites that are within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3’ terminal nucleotide of the guide region; a modified YA site at two or more guide region YA sites other than a 5’ end modification; or a modified YA site at a two or more guide region YA sites, wherein the modifications of the guide region YA sites comprise a modification at least one nucleotide located 5’ of the guide region YA site does not comprise.

[0149] In some other embodiments, the coRNA comprises: a modified YA site at three or more guide region YA sites that are at or after nucleotide 8 from the 5’ end of the 5’ terminus; a modified YA site at three or more guide region YA sites that are within nucleotides 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5’ end of the 5’ terminus; a modified YA site at three or more guide region YA sites that are within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3’ terminal nucleotide of the guide region; a modified YA site at three or more guide region YA sites other than a 5’ end modification; or a modified YA site at a three or more guide region YA sites, wherein the modifications of the guide region YA sites comprise a modification at least one nucleotide located 5’ of the guide region YA site does not comprise.

[0150] In some embodiments, the coRNA retains at least 75% of the activity of the parent coRNA.

[0151] In some embodiments of any of the aspects described herein, the nuclease is a transposon family nuclease. In some embodiments, the nuclease is a IS200 / IS605 transposon family encoded nuclease or a homolog or ortholog thereof. In some embodiments, the nuclease is ISC (insertion sequences Cas9-like), TnpB, or a homolog or ortholog thereof. In some embodiments, the nuclease is IscB nuclease, IsrB nuclease, TnpB nuclease, or a homolog or ortholog thereof, optionally, the nuclease is IscB nuclease or a homolog or ortholog thereof. In some embodiments, the nuclease is modified nuclease.

[0152] In some embodiments, the coRNA lacks from 1 to about 20 nucleotides present at 3 ’-end of the conserved portion of the parent coRNA.

[0153] In another aspect is a composition comprising a coRNA described herein or a polynucleotide encoding the same.

[0154] In some embodiments, the composition is in form of a lipid nanoparticles (LNP) or a protein nanoparticles (PNP).

[0155] In another aspect is a pharmaceutical composition comprising a coRNA described herein or a composition of any one of the preceding embodiments.

[0156] In another aspect is a DNA and RNA targeting system comprising: (i) a protein effector polypeptide or a polynucleotide encoding the protein effector polypeptide, optionally, the protein effector polypeptide has nuclease (e.g., endonuclease activity); and (ii) a coRNA of any one of the preceding embodiments or a polynucleotide encoding the coRNA.

[0157] In another aspect is a DNA and RNA targeting composition comprising: (i) a protein effector polypeptide or a polynucleotide encoding the protein effector polypeptide, optionally, theprotein effector polypeptide has nuclease (e.g., endonuclease activity); and (ii) a coRNA of any one of the preceding embodiments or a polynucleotide encoding the coRNA.

[0158] In some embodiments, the protein effector polypeptide comprises a RuvC domain, and / or an HNH domain. In some embodiments, the protein effector polypeptide comprises: (i) a split Ruv-C nuclease domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains; and / or (ii) an HNH domain. In some embodiments, the protein effector polypeptide comprises a PLMP domain and optionally a conserved C-terminal Y domain.

[0159] In some embodiments, the HNH domain is located in the RuvC domain (e.g., the HNH domain is located between RuvC-II and RuvC-III subdomains). In some embodiments, the protein effector polypeptide comprises a HNH domain but no RuvC domain. In some other embodiments, the protein effector polypeptide comprises a RuvC (e.g., split RuvC) domain but no HNH domain. In some embodiments, the protein effector polypeptide further comprises a REC domain. In some embodiments, the REC domain is downstream from the RuvC or HNH domain, however in some other embodiments, the REC domain is upstream from the RuvC or HNH domain. In some embodiments of any of the aspects described herein, the protein effector polypeptide comprises from about 170 to about 1000 amino acids.

[0160] In some embodiments, the coRNA comprises a guide sequence comprising a nucleotide sequence substantially complementary to at least a portion of a target nucleic acid (target sequence). In some embodiments, the target nucleic acid comprises a target adjacent motif (TAM) sequence 3’ of the target sequence. In some embodiments, the TAM comprises the sequence NAC, where N is A, C, G, or T.

[0161] In some embodiments, the DNA and RNA targeting system or composition further comprises a functional domain associated with the protein effector polypeptide. In some embodiments, the functional domain has transposase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, nucleic acid binding activity, detectable activity, or any combination thereof. In some preferred embodiments, the DNA and RNA targeting system or composition further comprises a serine or tyrosine recombinase.

[0162] In some embodiments, the DNA and RNA targeting system or composition further comprises a homologous recombination donor template comprising a donor sequence for insertion into a target polynucleotide.

[0163] In another aspect is an engineered cell comprising a DNA and RNA targeting system or composition of any one of the preceding embodiments.

[0164] In another aspect is a method of modifying a target nucleic acid, the method comprising contacting a DNA and RNA targeting system or composition of any one of the preceding embodiments.

[0165] In some embodiments, the target nucleic acid is in a cell and the method comprises administering to the cell a DNA and RNA targeting system or composition of any one of the preceding embodiments. In some embodiments, said modifying the target nucleic acid comprises cleaving the target nucleic acid. In some embodiments, said cleaving the target nucleic acid forms at least one 5’- overhang. In some embodiments, said modifying the target nucleic acid comprises deletion, insertion or change of a nucleotide in the target nucleic acid. In some embodiments, said modifying the target nucleic acid comprises editing (e.g., changing) one or more nucleobases; correcting or introducing a premature stop codon, disrupting a splice site; inserting or restoring a splice site, inserting a gene or gene fragment into the target nucleic acid, or a combination thereof.

[0166] In some embodiments of any of the aspects, the target nucleic acid is DNA. In some preferred embodiments, the target nucleic acid is genomic DNA, episomal DNA, viral DNA, or bacterial DNA. In some embodiments of any of the aspects, the target nucleic acid is double-stranded DNA. In some other embodiments of any of the aspects, the target nucleic acid is single-stranded DNA.

[0167] In some embodiments of any of the aspects, the target nucleic acid is RNA. In some other embodiments of any of the aspects, the target nucleic acid is double -stranded RNA. In some other embodiments, the target nucleic acid is single-stranded RNA. In some embodiments, the target nucleic acid is RNA selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmatic RNA (scRNA).

[0168] In some embodiments of any of the aspects described herein, the target nucleic acid is a disease- associated nucleic acid sequence.

[0169] In another aspect is an isolated nucleic acid modified by a method of anyone of the preceding embodiments.

[0170] In another aspect is an isolated cell or progeny thereof comprising a nucleic acid modified by a method of anyone of the preceding embodiments

[0171] Generally, the protein effector polypeptide and the guide nucleic acid described herein do not naturally occur together.BRIEF DESCRIPTION OF THE DRAWINGS

[0172] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0173] FIG. 1 examines the editing efficiency of engineered EboIscB variants over 48 genomic targets. The median editing efficiency over each variant is labeled. Wild-type EboIscB polypeptide is essentially pEDIT070.

[0174] FIG. 2A shows the engineering of TAMs either through the use of motif swaps or single mutants. FIG. 2B shows the validation of the human cell TAMs by examining the editing efficiency using either ATAAA targets or GTAAA targets.

[0175] FIGs. 3A-3C show strategies for engineering gRNA for efficiency and manufacturability. FIG. 3A examines the editing efficiency through the guide RNA: nuclease plasmid molar ratio. FIG. 3B shows nucleotides targeted for a shortened gRNA. FIG. 3C shows for three different targets that compact gRNA improves efficiency.

[0176] Figs 4A-4C examine secondary structure of OgeuIscB OmegaRNA and the predicted structure of WT and engineered EboIscB OmegaRNA. FIG. 4A depicts secondary folding structure of Wild-type OmegaRNA for OgeuIscB. A 20-nt variable region in each of the 5 ’-end of the OmegaRNA is labeled as “5’ N20”. For more details about the OgeuIscB polypeptide and associated OmegaRNA, see e.g., Kato et al. “Structure of the IscB-coRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9,” Nature Communications volume 13, Article number: 6719 (2022); and Altae-Tan et al., “The widespread IS200 / IS605 transposon family encodes diverse programmable RNA-guided endonucleases,” Science 2021 Oct, 374(6563): 57-65, the contents of which are incorporated herein by reference in their entirety; the contents of each of which are incorporated herein by reference in their entireties. FIG. 4B-4C show schematics of wild-type (FIG. 4B) or engineered M71 (FIG. 4C) OmegaRNA of EboIscB. Wild-type EboIscB polypeptide is essentially pEDIT070.DETAILED DESCRIPTION

[0177] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Protein effector polypeptides

[0178] Generally, the protein effector polypeptide has nuclease (e.g., endonuclease) activity. For example, the protein effector polypeptide described herein are RNase and have nuclease activity on RNA. In another example, the protein effector polypeptide described herein are DNase and have nuclease activity on DNA.

[0179] In some embodiments, the protein effector polypeptide is a transposon family encoded nuclease or a homolog or ortholog thereof. For example, the protein effector polypeptide is a IS200 / IS605 transposon family encoded nuclease or a homolog or ortholog thereof. In some embodiment, the protein effector polypeptide is IscB, Isrb, TnpB, or a homolog or ortholog thereof. Insome embodiments, the protein effector polypeptide is a modified IS200 / IS605 transposon family encoded nuclease.

[0180] In some embodiments, the protein effector polypeptide is a Class 2 CRISPR-Cas effector. Class 2 effectors contain an isolated CRISPR-Cas effector protein having a RuvC domain and an isolated RNA guide, comprising a tracrRNA and a crRNA, wherein the crRNA includes a spacer sequence that is complementary to a target nucleic acid sequence such as an RNA sequence. In some embodiments, the Class 2 CRISPR-Cas effector is derived from an uncultivated microorganism.

[0181] In some embodiments, the protein effector polypeptide is a class 2, type II Cas endonuclease. In Class 2 Type II systems there are two strands of RNA that are part of the CRISPR- Cas system: a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a complementary region of pre-crRNA facilitating maturation of the pre-crRNA to crRNA by an RNase III enzyme. The duplex formed by the tracrRNA and crRNA is recognized by, and associates with a protein, e.g., Cas9, which is directed to a target nucleic acid by a sequence of the crRNA that is complementary to, and hybridizes with, a sequence in the target nucleic acid. In some embodiments, the class 2, type II Cas endonuclease is not a Cas9 endonuclease, a Cas 14 endonuclease, a Cas 12a endonuclease, a Cas 12b endonuclease, a Cas 12c endonuclease, a Cas 12d endonuclease, a Casl2e endonuclease, a Casl3a endonuclease, a Cas 13b endonuclease, a Casl3c endonuclease, or a Cas 13d endonuclease. In some embodiments, the class 2, type II Cas endonuclease is derived from an uncultivated microorganism.

[0182] Class 2, type II Cas endonuclease comprise a RuvC domain and / or HNH domain. Accordingly, in some embodiments, the protein effector polypeptide described herein comprises one or more nuclease domains, such as RuvC domain and / or HNH domain. In some embodiments, the protein effector polypeptide comprises at least two nuclease domains, where one of the nuclease domains is responsible for the cleavage one strand of a double-stranded target nucleic acid and the other nuclease domains is responsible for cleavage of the other strand of the double-stranded target nucleic acid.

[0183] In some embodiments of any one of the aspects described herein, the protein effector polypeptide comprises a RuvC domain and / or a HNH domain. Without wishing to be bound by a theory, the RuvC domain is responsible for the cleavage of the target DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand. Generally, the protein effector polypeptide comprises at least one nuclease domain, i.e., the protein effector polypeptide comprises a RuvC domain or a HNH domain. In some embodiments, the protein effector polypeptide protein comprises at least two nuclease domains. For example, the protein effector polypeptide comprises at least two RuvC domains, or at least two HNH domains, or at least one RuvC domain and at least one HNH domain. In some preferred embodiments, the protein effector polypeptide comprises at least one RuvC domain and at least one HNH domain.

[0184] In some embodiments, the protein effector polypeptide is chimeric. For example, the protein effector polypeptide comprises a first fragment and a second fragment, and wherein the fragments can be of protein effector polypeptide orthologs of organisms of a genus or of species. In some embodiments, the protein effector polypeptide comprises a first fragment and a second fragment, and wherein the first and second fragments are from protein effector polypeptide orthologs of different species.

[0185] In some embodiments, the protein effector polypeptide is an modified IscB comprising the sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any one of the sequences:MRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTLPFTIQLTYKPRTRI LQPVTLGQDPGRTNIGMAAVRFDGKELGRFHCITRNKEIPKLMADRMAARKA SRRGERLARKRLARKLHTTAKHLNGRILPGCSEPIAVKDIINTESRFNNRLRPE GWLTPTATQLLRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKREID FCHGPLCGTGGLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIANIA GLCPKCHELVHKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVETLADLFP GHFHVTNGWNTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFEILQF RKHNRAIIHHQTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAKEHG KTQADAMRSRLTVIKSTRYYNTPGRMMPGTVFLYEGKRYVMTGQITNGKYY RAYGQEKRNFPAVKVRILTKNTGLVFVA (SEQ ID NO: 1; pEDIT070);MRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTLPFTIQLTYKPRTRI LQPVTLGQDPGRTNIGMAAVRFDGKELGRFHCITRNKEIPKLMADRMAARKA SRRGERLARKRLARKLHTTAKHLNGRILPGCSEPIAVKDIINTESRFNNRILTKC KVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEGQQGNINKLFRKL KFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIEKSTFRLTPTATQL LRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKREIDFCHGPLCGTG GLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIANIAGLCPKCHELV HKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVETLADLFPGHFHVTNGW NTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFEILQFRKHNRAIIHH QTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAKEHGKTQADAMRS RLTVIKSTRYYNTPGRMMPGTVFLYEGKRYVMTGQITNGKYYRAYGQEKRNFPAVKVRILTKNTGLVFVA (SEQ ID NO: 2; pEDIT071);MRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTLPFTIQLTYKPRTRI LQPVTLGQDPGRTNIGMAAVRFDGKELGRFHCITRNKEIPKLMADRMAARKA SRRGERLARKRLARKLHTTAKHLNGRILPGCSKPIAVKDIINTESRFNNRILTKC KVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEGQQGNINKLFRKL KFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIEKSTFRLTPTATQL LRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKREIDFCHGPLCGTG GLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIANIAGLCPKCHELV HKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVRTLAKLFPGHFHVTNGWNTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFEILQFRKHNRAIIHH QTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAKEHGKTQADAMRS RLTVKKSRRYYNTPGRMMPGTVFLYEGKRYVMTGQITNGKYYRAYGQEKRN FPAVKVRILTKNTGLVFVA (SEQ ID NO: 3; pEDIT416);MRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTLPFTIQLTYKPRTRI LQPVTLGQDPGRTHIGMAAVRFDGKELGRFHCITRNKEIPKLMADRMKARKA SRRGERLARKRLARKLHTTAKHLNGRILPGCSKPIAVKDIINTESRFNNRILTKC KVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEGQQGNINKLFRKL KFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIEKSTFRLTPTATQL LRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKREIDFCHGPLCGTG GLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIANIAGLCPKCHELV HKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVRTLAKLFPGHFHVTNGW NTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFEILQFRKHNRAIIHH QTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAKEHGKTQADAMRS RLTVKKSTRYYNTPGRMMPGTVFLYEGKRYVMTGQITNGKYYRAYGQEKRN FPAVKVRILTKNTGLVFVA (SEQ ID NO: 4 pEDIT417);MRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTLPFTIQLTYKPRTRI LQPVTLGQDPGRTHIGMAAVRFDGKELGRFHCITRNKEIPKLMADRMAARKA SRRGERLARKRLARKLHTTAKHLNGRILPGCSKPIAVKDIINTESRFNNRILTKC KVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEGQQGNINKLFRKL KFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIEKSTFRLTPTATQL LRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKREIDFCHGPLCGTG GLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIANIAGLCPKCHELV HKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVRTLAKLFPGHFHVTNGW NTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFEILQFRKHNRAIIHH QTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAKEHGKTQADAMRS RLTVKKSRRYYNTPGRMMPGTVFLYEGKRYVMTGQITNGKYYRAYGQEKRN FPAVKVRILTKNTGLVFVA (SEQ ID NO: 5; pEDIT418);MGSPKKKRKVGSMRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTL PFTIQLTYKPRTRILQPVTLGQDPGRTNIGMAAVRFDGKELGRFHCITRNKEIPK LMADRMAARKASRRGERLARKRLARKLHTTAKHLNGRILPGCSEPIAVKDIIN TESRFNNRILTKCKVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEG QQGNINKLFRKLKFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIE KSTFRLTPTATQLLRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKR EIDFCHGPLCGTGGLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIA NIAGLCPKCHELVHKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVRTLA KLFPGHFHVTNGWNTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFE ILQFRKHNRAIIHHQTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAK EHGKTQADAMRSRLTVKKSTRYYNTPGRMMPGTVFLYEGKRYVMTGQITNG KYYRAYGQEKRNFPAVKVRILTKNTGLVFVAGSKRPAATKKAGQAKKKKGS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 6; pEDIT0152, triple- mutant); orMGSPKKKRKVGSMRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTL PFTIQLTYKPRTRILQPVTLGQDPGRTHIGMAAVRFDGKELGRFHCITRNKEIPK LMADRMKARKASRRGERLARKRLARKLHTTAKHLNGRILPGCSKPIAVKDIIN TESRFNNRILTKCKVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEG QQGNINKLFRKLKFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIEKSTFRLTPTATQLLRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKR EIDFCHGPLCGTGGLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIA NIAGLCPKCHELVHKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVRTLA KLFPGHFHVTNGWNTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFE ILQFRKHNRAIIHHQTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAK EHGKTQADAMRSRLTVKKSRRYYNTPGRMMPGTVFLYEGKRYVMTGQITNG KYYRAYGQEKRNFPAVKVRILTKNTGLVFVAGSKRPAATKKAGQAKKKKGS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 7; pEDIT0420; 7-mutant).MGSPKKKRKVGSMRYVYVLDVDGKPLMPTCRFGKVRRMLKSGQAKAVDTL PFTIQLTYKPRTRILQPVTLGQDPGRTNIGMAAVRFDGKELGRFHCITRNKEIPK LMADRMAARKASRRGERLARKRLARKLHTTAKHLNGRILPGCSEPIAVKDIIN TESRFNNRILTKCKVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEG QQGNINKLFRKLKFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIE KSTFRLTPTATQLLRTHINLFRKLSGILPVTDVAVELNKFAFMQLDNPEMKKR EIDFCHGPLCGTGGLEAAVKEQQDGKCLLCGKESIGHYHHIVPRSRRGSNTIA NIAGLCPKCHELVHKDADTAESLTEMKTGLMKKYGGTSVLNQIIPKLVETLAD LFPGHFHVTNGWNTKEFREKHHLEKDHDVDAYCIACSHLKPEETLVETEPFEI LQFRKHNRAIIHHQTERTYKLDGVTVAKNRKKRMEQKTDSLEDWYVDMAKE HGKTQADAMRSRLTVIKSTRYYNTPGRMMPGTVFLYEGKRYVMTGQITNGK YYRAYGQEKRNFPAVKVRILTKNTGLVFVA (SEQ ID NO: 8; pEDIT071+NLS)

[0186] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine of positions 18, 34, 36, 37, 60, 66, 85, 97, 99, 100, 101, 104, 109, 112, 118, 136, 137, 155, 156, 157, 166, 168, 190, 204, 204, 205, 206, 208, 212, 214, 216, 218,219, 229, 229, 230, 233, 233, 234, 234, 238, 239, 241, 242, 245, 246, 248, 252, 254, 255, 256, 257,258, 279, 280, 291, 293, 298, 303, 304, 306, 307, 312, 319, 323, 328, 329, 331, 332, 338, 341, 353,360, 368, 372, 376, 377, 378, 379, 380, 381, 382, 384, 385, 386, 388, 390, 391, 393, 405, 406, 408,409, 410, 412, 413, 416, 417, 418, 419, 421, 421, 423, 425, 437, 439, 455, 462, 463, 478, 479, 533,536, 564 and 565 of the amino acid sequence of SEQ ID NO: 2 (pEDIT071). Optionally, if the protein effector polypeptide comprises the mutation Q60I, N66H, I85R, I85K, E137R, P405K, E409K, E409R, or D413R, the protein effector polypeptide comprises a mutation at least one other position from the above and / or a mutation other than Q60I, N66H, I85R, I85K, E137R, P405K, E409K, E409R, or D413R. Without wishing to be bound by a theory, protein effector polypeptide comprises an amino acid sequence comprising a mutation at one or more of the above positions have higher or increased gene-editing efficiency than the protein effector polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protein effector polypeptide comprising a mutation at one or more of the above listed positions has at least 12-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 10-fold, at least 15-fold, at least 20-fild, at least 25-fold, at least 30-fold, at least 35 fold, at least 40-fold, at least 45 fold, or at least 50-fold higher editing efficiency than the protein effector polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

[0187] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of variant C18R, A34R, D36R, T37R, Q60I, N66H, I85X, I85X, M99X, A101X, A104R, E109X, A112K, R118K, S136X, E137R, I155X, L156X, T157X, T166X, L168K, L190K, G204X, N205X, I206K, K208R, K212R, K214R, N216R, K218R, D219X, A229X, K230R, L233X, P234X, P238X, F239X, K241R, E242X, A245X, E246X, E248R, T252X, E254X, K255R, S256X, T257X, F258X, S279R, G280R, L291X, K293R, Q298K, E303X, M304K, K306R, R307K, C312X, T319K, E323X, E328R, Q329R, D331R, G332R, G338R, S341R, R353K, A360X, K368R, L372R, D376R, A377R, D378R, T379R, A380R, E381R, S382R, T384R, E385R, M386R, T388R, L390X, M391X, K393R, P405K, K406R, V408R, E409X, T410R, A412R, D413R, P416R, G417R, H418K, F419R, V421X, N423R, W425K, E437R, D439X, E455R, E462R, P463R, H478X, H479X, I564X, and T565X of the amino acid sequence provided in SEQ ID NO: 2, wherein X is any amino acid. Optionally, if the protein effector polypeptide comprises the mutation Q60I, N66H, I85R, I85K, E137R, P405K, E409K, E409R, or D413R, the protein effector polypeptide comprises a mutation at least one other position from the above and / or a mutation other than Q60I, N66H, I85R, I85K, E137R, P405K, E409K, E409R, or D413R.

[0188] Some exemplary specific mutations of SEQ ID NO: 2 are shown in Table A.Table A: Exemplary positions and mutations for SEQ ID NO: 2

[0189] Some exemplary specific mutations of SEQ ID NO: 8 or SEQ ID NO: 7 are shown in TableA2.Table A2: Exemplary positions and mutations for SEQ ID NO: 8 or SEQ ID NO: 7.

[0190] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of variant C18R, A34R, D36R, T37R, Q60I, N66H, I85X, I85X, M99X, A101X, A104R, E109X, A112K, R118K, S136X, E137R, I155X, L156X, T157X, T166X, L168K, L190K, G204X, N205X, I206K, K208R, K212R, K214R, N216R, K218R, D219X, A229X, K230R, L233X, P234X, P238X, F239X, K241R, E242X, A245X, E246X, E248R, T252X, E254X, K255R, S256X, T257X, F258X, S279R, G280R, L291X, K293R, Q298K, E303X, M304K, K306R, R307K, C312X, T319K, E323X, E328R, Q329R, D331R, G332R, G338R, S341R, R353K, A360X, K368R, L372R, D376R, A377R, D378R, T379R, A380R, E381R, S382R, T384R, E385R, M386R, T388R, L390X, M391X, K393R, P405K, K406R, V408R, E409X, T410R, A412R, D413R, P416R, G417R, H418K, F419R, V421X, N423R, W425K, E437R, D439X, E455R, E462R, P463R, H478N, H479N, I564K, I564H, I564N, I564Q, I564C, I564M, I564T, I564D, T565R, T565K, T565H, T565C, T565D, T565G, H478C, H478P, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 1, wherein X is any amino acid. Optionally, if the protein effector polypeptide comprises the mutation Q60I, N66H, I85R, I85K, E137R, P405K, E409K, E409R, or D413R, the protein effector polypeptide comprises a mutation at least one other position from the above and / or a mutation other than Q60I, N66H, I85R, I85K, E137R, P405K, E409K, E409R, or D413R.

[0191] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at least one, at least two, at least three, at least four, at least five, at least six, or at least seven of positions 409, 413, 533, 66, 100, 137 and 536 of the amino acid sequence of SEQ IDNO: 2. Optionally, if the protein effector polypeptide comprises the mutation N66H, I85R, I85K, E137R, P405K, E409K, E409R, or D413R, the protein effector polypeptide comprises a mutation at least one other position from the above and / or a mutation other than N66H, E 137K, E409K, E409R, or D413R, the protein effector polypeptide comprises a mutation at least one (e.g., at least two, at least three, at least four, at least five or more) other position (e.g., at position 18, 34, 36, 37, 60, 85, 97, 99, 100, 101, 104, 109, 112, 118, 136, 155, 156, 157, 166, 168, 190, 204, 204, 205, 206, 208, 212, 214,216, 218, 219, 229, 229, 230, 233, 233, 234, 234, 238, 239, 241, 242, 245, 246, 248, 252, 254, 255,256, 257, 258, 279, 280, 291, 293, 298, 303, 304, 306, 307, 312, 319, 323, 328, 329, 331, 332, 338,341, 353, 360, 368, 372, 376, 377, 378, 379, 380, 381, 382, 384, 385, 386, 388, 390, 391, 393, 405,406, 408, 410, 412, 416, 417, 418, 419, 421, 421, 423, 425, 437, 439, 455, 462, 463, 533, or 536) and / or a mutation other than N66H, E137K, E409K, E409R, or D413R.

[0192] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 409, 413, and 533 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, and I533R in the amino acid sequence of SEQ ID NO: 2. Without wishing to be bound by a theory, protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 409, 413, and 533 in the amino acid sequence of SEQ ID NO: 2 have higher or increased gene-editing efficiency than the protein effector polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protein effector polypeptide comprising mutations at least at positions 409, 413, and 533 has at least 12-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 10-fold, at least 15 -fold, at least 20-fild, at least 25 -fold, at least 30-fold, at least 35 fold, at least 40-fold, at least 45 fold, or at least 50-fold higher editing efficiency than the protein effector polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

[0193] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 421, 425, and 545 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D425K, and I545R in the amino acid sequence of SEQ ID NO: 8. Without wishing to be bound by a theory, protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 421, 425, and 545 in the amino acid sequence of SEQ ID NO: 8 have higher or increased gene-editing efficiency than the protein effector polypeptide comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the protein effector polypeptide comprising mutations at least at positions 421, 425, and 545 has at least 12-fold, at least 3-fold, at least 4-fold, at least 5 -fold, at least 10-fold, at least 15 -fold, at least 20-fild, at least 25 -fold, at least 30-fold, at least 35 fold, at least 40-fold, at least 45 fold, or at least 50-fold higher editing efficiency than the protein effector polypeptide comprising the amino acid sequence of SEQ ID NO: 8.Triple + modifications

[0194] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 409, 413, and 533, and a mutation at least one (e.g., two, three or four) of position 66, 100, 137 536 of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K and I533R, and at least one (e.g., two, three, or four) of mutations N66H, A100K, E137K and T536R in the amino acid sequence of SEQ ID NO: 2.

[0195] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 409, 413, 533, and 66 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, and N66H in the amino acid sequence of SEQ ID NO: 2.

[0196] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 409, 413, 533, and 100 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, and A100K in the amino acid sequence of SEQ ID NO: 2.

[0197] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 409, 413, 533, and 137 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, and E137K in the amino acid sequence of SEQ ID NO: 2.

[0198] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, and T536R in the amino acid sequence of SEQ ID NO: 2.

[0199] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 66, and 100 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, N66H, and A100K in the amino acid sequence of SEQ ID NO: 2.

[0200] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 66, and 137 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, N66H, and E137K in the amino acid sequence of SEQ ID NO: 2

[0201] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 66, and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, N66H, and T536R in the amino acid sequence of SEQ ID NO: 2.

[0202] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 100, and 137 in the amino acid sequence of SEQ ID NO: 2. For example, theprotein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, A100K, and E137 in the amino acid sequence of SEQ ID NO: 2.

[0203] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 100, and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, A100K, and T536R in the amino acid sequence of SEQ ID NO: 2.

[0204] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 137 and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, E137K and T536R in the amino acid sequence of SEQ ID NO: 2.

[0205] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 66, 100, and 137 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, N66H, A100K, and E137K in the amino acid sequence of SEQ ID NO: 2.

[0206] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 66, 100, and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, N66H, A100K, and T536R in the amino acid sequence of SEQ ID NO: 2.

[0207] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 66, 137 and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, N66H, E137K and T536R in the amino acid sequence of SEQ ID NO: 2.

[0208] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 100, 137 and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, A100K, E137K and T536R in the amino acid sequence of SEQ ID NO: 2.

[0209] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 409, 413, 533, 66, 100, 137 and 536 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E409K, D413K, I533R, N66H, A100K, E137K and T536R in the amino acid sequence of SEQ ID NO: 2.

[0210] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 421, 423, and 545, and a mutation at least one (e.g., two, three or four) of position 78, 112, 149 548 of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K and I545R, and at leastone (e.g., two, three, or four) of mutations N78H, A112K, E149K and T548R in the amino acid sequence of SEQ ID NO: 8.

[0211] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 421, 423, 545, and 78 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, and N78H in the amino acid sequence of SEQ ID NO: 8.

[0212] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at positions 421, 423, 545, and 112 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, and Al 12K in the amino acid sequence of SEQ ID NO: 8.

[0213] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 421, 423, 545, and 149 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, and E149K in the amino acid sequence of SEQ ID NO: 8.

[0214] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, and 548 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, and T548R in the amino acid sequence of SEQ ID NO: 8.

[0215] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 78, and 112 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, N78H, and Al 12K in the amino acid sequence of SEQ ID NO: 8.

[0216] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 78, and 149 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, N78H, and E149K in the amino acid sequence of SEQ ID NO: 8

[0217] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 78, and 548 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, N78H, and T548R in the amino acid sequence of SEQ ID NO: 8.

[0218] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 112, and 149 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, Al 12K, and E149 in the amino acid sequence of SEQ ID NO: 8.

[0219] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 112, and 548 in the amino acid sequence of SEQ ID NO: 8. For example, theprotein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, Al 12K, and T548R in the amino acid sequence of SEQ ID NO: 8.

[0220] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 149 and 548 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, E149K and T548R in the amino acid sequence of SEQ ID NO: 8.

[0221] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 78, 112, and 149 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, N78H, Al 12K, and E149K in the amino acid sequence of SEQ ID NO: 8.

[0222] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 78, 112, and 548 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, N78H, Al 12K, and T548R in the amino acid sequence of SEQ ID NO: 8.

[0223] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 78, 149 and 548 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, N78H, E149K and T548R in the amino acid sequence of SEQ ID NO: 8.

[0224] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 112, 149 and 548 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, Al 12K, E149K and T548R in the amino acid sequence of SEQ ID NO: 8.

[0225] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising 421, 423, 545, 78, 112, 149 and 548 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising the mutations E421K, D423K, I545R, N78H, Al 12K, E149K and T548R in the amino acid sequence of SEQ ID NO: 8.Stacking of mutations

[0226] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising a mutation at positions 409, 413, and 533, and at least one other mutation at a position listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K and I533R, and at least one other mutation listed in Table A.

[0227] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising a mutation at positions 409, 413, 533 and 66, and at least one othermutation at a position listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, and N66H, and at least one other mutation listed in Table A.

[0228] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising a mutation at positions 409, 413, 533, and 100, and at least one other mutation at a position listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, and A100K, and at least one other mutation listed in Table A.

[0229] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising a mutation at positions 409, 413, 533, and 137, and at least one other mutation at a position listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, and E137K, and at least one other mutation listed in Table A.

[0230] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, and T536R, and at least one other mutation listed in Table A.

[0231] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 66, and 100, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, N66H, and A100K, and at least one other mutation listed in Table A.

[0232] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 66, and 137, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, N66H, and E137K, and at least one other mutation listed in Table A.

[0233] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 66, and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, N66H, and T536R, and at least one other mutation listed in Table A.

[0234] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 100, and 137, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ IDNO: 2 and comprising the mutations E409K, D413K, I533R, A100K, and E137, and at least one other mutation listed in Table A.

[0235] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 100, and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, A100K, and T536R, and at least one other mutation listed in Table A.

[0236] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 137 and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, E137K and T536R, and at least one other mutation listed in Table A.

[0237] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 66, 100, and 137, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, N66H, A100K, and E137K, and at least one other mutation listed in Table A.

[0238] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 66, 100, and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, N66H, A100K, and T536R, and at least one other mutation listed in Table A.

[0239] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 66, 137 and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, N66H, E137K and T536R, and at least one other mutation listed in Table A.

[0240] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 100, 137 and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, A100K, E137K and T536R, and at least one other mutation listed in Table A.

[0241] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 2 and comprising 409, 413, 533, 66, 100, 137 and 536, and at least one other mutation listed in Table A. For example, the protein effector polypeptide comprises an amino acid sequence ofSEQ ID NO: 2 and comprising the mutations E409K, D413K, I533R, N66H, A100K, E137K and T536R, and at least one other mutation listed in Table A.

[0242] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising a mutation at positions 421, 423, and 545, and at least one other mutation at a position listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K and I545R, and at least one other mutation listed in Table A2.

[0243] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising a mutation at positions 421, 423, 545 and 78, and at least one other mutation at a position listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, and N78H, and at least one other mutation listed in Table A2.

[0244] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising a mutation at positions 421, 423, 545, and 112, and at least one other mutation at a position listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, and Al 12K, and at least one other mutation listed in Table A2.

[0245] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising a mutation at positions 421, 423, 545, and 149, and at least one other mutation at a position listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, and E149K, and at least one other mutation listed in Table A2.

[0246] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E42 IK, D423K, I545R, and T548R, and at least one other mutation listed in Table A2.

[0247] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 78, and 112, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, N78H, and Al 12K, and at least one other mutation listed in Table A2.

[0248] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 78, and 149, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ IDNO: 8 and comprising the mutations E421K, D423K, I545R, N78H, and E149K, and at least one other mutation listed in Table A2.

[0249] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 78, and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, N78H, and T548R, and at least one other mutation listed in Table A2.

[0250] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 112, and 149, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, Al 12K, and E149, and at least one other mutation listed in Table A2.

[0251] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 112, and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, Al 12K, and T548R, and at least one other mutation listed in Table A2.

[0252] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 149 and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, E149K and T548R, and at least one other mutation listed in Table A2.

[0253] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 78, 112, and 149, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, N78H, A112K, and E149K, and at least one other mutation listed in Table A2.

[0254] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 78, 112, and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, N78H, A112K, and T548R, and at least one other mutation listed in Table A2.

[0255] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 78, 149 and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQID NO: 8 and comprising the mutations E421K, D423K, I545R, N78H, E149K and T548R, and at least one other mutation listed in Table A2.

[0256] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 112, 149 and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, A112K, E149K and T548R, and at least one other mutation listed in Table A2.

[0257] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising 421, 423, 545, 78, 112, 149 and 548, and at least one other mutation listed in Table A2. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID NO: 8 and comprising the mutations E421K, D423K, I545R, N78H, A112K, E149K and T548R, and at least one other mutation listed in Table A2.Mutations for TAM variants

[0258] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at one, two, three or four of positions 478, 479, 564 and 565 of the amino acid of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at least at positions 478 and 479, at positions 478 and 565, at positions 478 and 565, at positions 479 and 564, at positions 479 and 565, at positions 565 and 565, at positions 478, 479 and 564, at positions 478, 479 and 565, at positions 478, 564 and 565, at positions 479, 564 and 565, or at positions 478, 479, 564 and 565 of SEQ ID NO: 2.

[0259] It is noted that protein effector polypeptide comprising mutations at one, two, three or four of positions 478, 479, 564 and 565 of the amino acid of SEQ ID NO: 2 exhibit increased activity on a target sequence comprising a non-canonical TMA sequence. They are particularly effective on target sequences comprising the TAM sequence RTR, where R is purine (e.g., G or A). In some embodiments, the protein effector polypeptide comprising mutations at one, two, three or four of positions 478, 479, 564 and 565 of the amino acid of SEQ ID NO: 2 exhibit increased activity on a target sequence comprising the TAM sequence ATR. Particularly, the protein effector polypeptide comprising mutations at one, two, three or four of positions 478, 479, 564 and 565 of the amino acid of SEQ ID NO: 2 exhibit increased activity on a target sequence comprising the TAM sequence ATR in human cells.

[0260] In some embodiments, the t protein effector polypeptide comprising mutations at one, two, three or four of positions 478, 479, 564 and 565 of the amino acid of SEQ ID NO: 2 exhibits increased activity on a target sequence comprising a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM,ATRRM, ATRMA, ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR (where R is purine (e.g., A or G) and M is A) at its 3' end as compared to SEQ ID NO: 2.

[0261] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 478 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising a H478N, H478C or H479P mutation in the amino acid sequence of SEQ ID NO: 2. In some preferred embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a H478N mutation in the amino acid sequence of SEQ ID NO: 2.

[0262] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 479 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising a H479N, H479C or H479M mutation in the amino acid sequence of SEQ ID NO: 2. In some preferred embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a H479N mutation in the amino acid sequence of SEQ ID NO: 2.

[0263] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 564 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising a I564R, I564K, I564H, I564N, I564Q, I564C, I564C, I564T, or I564D mutation in the amino acid sequence SEQ ID NO: 2. In some preferred embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a I564R mutation in the amino acid sequence of SEQ ID NO: 2.

[0264] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 565 in the amino acid sequence of SEQ ID NO: 2. For example, the protein effector polypeptide comprises an amino acid sequence comprising a T565R, T565K, T564H, T565C, T565D, or T565G mutation in the amino acid sequence of SEQ ID NO: 2. In some preferred embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a T565R mutation in the amino acid sequence of SEQ ID NO: 2.

[0265] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at one, two, three or four of positions 490, 491, 576 and 577 of the amino acid of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at least at positions 490 and 491, at positions 490 and 577, at positions 490 and 577, at positions 491 and 576, at positions 491 and 577, at positions 577 and 577, at positions 490, 491 and 576, at positions 490, 491 and 577, at positions 490, 576 and 577, at positions 491, 576 and 577, or at positions 490, 491, 576 and 577 of SEQ ID NO: 8.

[0266] It is noted that protein effector polypeptide comprising mutations at one, two, three or four of positions 490, 491, 576 and 577 of the amino acid of SEQ ID NO: 8 exhibit increased activity on a target sequence comprising a non-canonical TMA sequence. They are particularly effective on targetsequences comprising the TAM sequence RTR, where R is purine (e.g., G or A). In some embodiments, the protein effector polypeptide comprising mutations at one, two, three or four of positions 490, 491, 576 and 577 of the amino acid of SEQ ID NO: 8 exhibit increased activity on a target sequence comprising the TAM sequence ATR. Particularly, the protein effector polypeptide comprising mutations at one, two, three or four of positions 490, 491, 576 and 577 of the amino acid of SEQ ID NO: 8 exhibit increased activity on a target sequence comprising the TAM sequence ATR in human cells.

[0267] In some embodiments, the t protein effector polypeptide comprising mutations at one, two, three or four of positions 490, 491, 576 and 577 of the amino acid of SEQ ID NO: 8 exhibits increased activity on a target sequence comprising a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM, ATRRM, ATRMA, ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR (where R is purine (e.g., A or G) and M is A) at its 3' end as compared to SEQ ID NO: 8.

[0268] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 490 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising a H490N, H490C or H49 IP mutation in the amino acid sequence of SEQ ID NO: 8. In some preferred embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a H490N mutation in the amino acid sequence of SEQ ID NO: 8.

[0269] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 491 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising a H49 IN, H491C or H491M mutation in the amino acid sequence of SEQ ID NO: 8. In some preferred embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a H49 IN mutation in the amino acid sequence of SEQ ID NO: 8.

[0270] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 576 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising a I576R, I576K, I576H, I576N, I576Q, I576C, I576C, I576T, or I576D mutation in the amino acid sequence SEQ ID NO: 8. In some preferred embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a I576R mutation in the amino acid sequence of SEQ ID NO: 8.

[0271] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising a mutation at position 577 in the amino acid sequence of SEQ ID NO: 8. For example, the protein effector polypeptide comprises an amino acid sequence comprising a T577R, T577K, T576H, T577C, T577D, or T577G mutation in the amino acid sequence of SEQ ID NO: 8. In some preferredembodiments, the protein effector polypeptide comprises an amino acid sequence comprising a T577R mutation in the amino acid sequence of SEQ ID NO: 8.

[0272] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID No: 2, where at least one, at least two, at least three, at last four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten, at least eleven amino acid sequence between positions 560 to 571 is changed. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID No: 2, where at least one, at least two, at least three, at last four, at least five, or at least six amino acid sequence between positions 563 to 568 is changed.

[0273] Without wishing to be bound by a theory, protein effector polypeptides comprising a one, two, three, four five or six amino acid sequence swap at positions 563 to 568, or a one, two, three, four five, six, seven, eight, nine, ten or eleven amino acid sequence swap at position 560-571 of SEQ ID NO: 2 exhibit increased activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A).

[0274] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising the sequence QHCHGA, NSHYGQ, QHCHGN, QQNKGD, THAKS, QKNNGV, VLNKGA, SSGAGK, NSNGGA, IKNRGR, MHNKGK, VFNYGK, THNKGA, AHGYGK, NKNKGK, THSYGK, MHSYGR, SQNKGT, TTGLGK, KTNN, TQNKGR, THCKGT, QHTNGR, QRTNGN, QRTNGS, QRTRGQ, QASGGK, QSSGGK, QQSNGR, QHSNGA, QKNRGY, QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, QLSKGI, QKDKGA, TKNKGT, or GKNGYV at position Q563-K568 of SEQ ID NO: 2.

[0275] Without wishing to be bound by a theory, protein effector polypeptides comprising the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, and QKDKGA at position 563-568 of SEQ ID NO: 2 exhibit increased activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A). Accordingly, in some embodiments, the protein polypeptide comprises the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, and QKDKGA at position 563-568 of SEQ ID NO: 2, and wherein the protein effector polypeptide has editing activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A) that is higher than the editing activity of protein effector polypeptide comprising the SEQ ID NO: 2. For example, the protein polypeptide comprising the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, and QKDKGA at position 563-568 of SEQ ID NO: 2 has higher editing activity compared to the editing activity of protein effector polypeptide comprising the SEQ ID NO: 2 when the target sequence comprises a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM, ATRRM, ATRMA, ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR (where R is purine (e.g., A or G) and M is A) at its 3' end.

[0276] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising the sequence MSGQHCHGAYLR, LSGNSHYGQYYM, MTGQHCHGNYFR,MTGQQNKGDRYF, KSGQLTNGQYFR, LKGNACNGAYFH, SSGTHAKSYFR,LGGQKNNGVYFY, VTGSSGAGKYVR, VRSVHSYGKYVR, VQGIKNRGRYLH,LRGTSSYGKTVL, VKGVFNYGKWVR, VKGKHNDRWI, VKGVQNRGKYIK,IKGSHCNGSRLI, SGGVMSYGRYIT, SKGNKNKGKSIL, AIGTHCKGKRVI,VKGVFNRGTWVR, CKGMHSYGRYVL, IGGTQNKGRYAI, or TKGTHCKGTRIM at position M560-R571 of SEQ ID NO: 2.

[0277] Without wishing to be bound by a theory, protein effector polypeptides comprising one of these sequences at position 560-571 of SEQ ID NO: 2 exhibit increased activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A). Accordingly, in some embodiments, the protein polypeptide comprises the one of the above sequences at position 650-571 of SEQ ID NO: 2, and wherein the protein effector polypeptide has editing activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A) that is higher than the editing activity of protein effector polypeptide comprising the SEQ ID NO: 2. For example, the protein polypeptide comprising the one of the above sequences at position 560-571 of SEQ ID NO: 2 has higher editing activity compared to the editing activity of protein effector polypeptide comprising the SEQ ID NO: 2 when the target sequence comprises a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM, ATRRM, ATRMA, ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR (where R is purine (e.g., A or G) and M is A) at its 3' end.

[0278] In some embodiments, the protein effector polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a protein effector polypeptide sequence shown in Table B.

[0279] Table B: Some exemplary protein effector polypeptide sequences!II1

[0280] In some embodiments, the protein effector polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with an homolog or ortholog of a protein effector polypeptide sequence shown in Table B.

[0281] Some exemplary protein effector polypeptides comprising a RuvC and / or HNH domain are described, for example, in PCT publications WO2022087494, W02020180699, WO2021226363, WO2022261292, WO2021119275, WO2019237069, W02018107028, WO2019067872 and WO2019067992, content of each of which is incorporated herein by reference in their entireties.

[0282] In some embodiments, the protein effector polypeptide comprises an amino acid sequence of SEQ ID No: 8, where at least one, at least two, at least three, at last four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten, at least eleven amino acid sequence between positions 572 to 583 is changed. For example, the protein effector polypeptide comprises an amino acid sequence of SEQ ID No: 8, where at least one, at least two, at least three, at last four, at least five, or at least six amino acid sequence between positions 575 to 580 is changed.

[0283] Without wishing to be bound by a theory, protein effector polypeptides comprising a one, two, three, four five or six amino acid sequence swap at positions 575 to 580, or a one, two, three, four five, six, seven, eight, nine, ten or eleven amino acid sequence swap at position 572-583 of SEQ ID NO: 8 exhibit increased activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A).

[0284] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising the sequence QHCHGA, NSHYGQ, QHCHGN, QQNKGD, THAKS, QKNNGV, VLNKGA, SSGAGK, NSNGGA, IKNRGR, MHNKGK, VFNYGK, THNKGA, AHGYGK,NKNKGK, THSYGK, MHSYGR, SQNKGT, TTGLGK, KTNN, TQNKGR, THCKGT, QHTNGR, QRTNGN, QRTNGS, QRTRGQ, QASGGK, QSSGGK, QQSNGR, QHSNGA, QKNRGY, QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, QLSKGI, QKDKGA, TKNKGT, or GKNGYV at position Q575-K580 of SEQ ID NO: 8.

[0285] Without wishing to be bound by a theory, protein effector polypeptides comprising the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, and QKDKGA at position 575-580 of SEQ ID NO: 8 exhibit increased activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A). Accordingly, in some embodiments, the protein polypeptide comprises the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, and QKDKGA at position 575-580 of SEQ ID NO: 8, and wherein the protein effector polypeptide has editing activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A) that is higher than the editing activity of protein effector polypeptide comprising the SEQ ID NO: 8. For example, the protein polypeptide comprising the sequence QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, and QKDKGA at position 575-580 of SEQ ID NO: 8 has higher editing activity compared to the editing activity of protein effector polypeptide comprising the SEQ ID NO: 8 when the target sequence comprises a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM, ATRRM, ATRMA,ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR (where R is purine (e.g., A or G) and M is A) at its 3' end.

[0286] In some embodiments, the protein effector polypeptide comprises an amino acid sequence comprising the sequence MSGQHCHGAYLR, LSGNSHYGQYYM, MTGQHCHGNYFR,MTGQQNKGDRYF, KSGQLTNGQYFR, LKGNACNGAYFH, SSGTHAKSYFR,LGGQKNNGVYFY, VTGSSGAGKYVR, VRSVHSYGKYVR, VQGIKNRGRYLH,LRGTSSYGKTVL, VKGVFNYGKWVR, VKGKHNDRWI, VKGVQNRGKYIK,IKGSHCNGSRLI, SGGVMSYGRYIT, SKGNKNKGKSIL, AIGTHCKGKRVI,VKGVFNRGTWVR, CKGMHSYGRYVL, IGGTQNKGRYAI, or TKGTHCKGTRIM at positionM572-R583 of SEQ ID NO: 8.

[0287] Without wishing to be bound by a theory, protein effector polypeptides comprising one of these sequences at position 572-583 of SEQ ID NO: 8 exhibit increased activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A). Accordingly, in some embodiments, the protein polypeptide comprises the one of the above sequences at position 650-583 of SEQ ID NO: 8, and wherein the protein effector polypeptide has editing activity on a target sequence comprising the TAM sequence RTAAA, where R is purine (e.g., G or A) that is higher than the editingactivity of protein effector polypeptide comprising the SEQ ID NO: 8. For example, the protein polypeptide comprising the one of the above sequences at position 572-583 of SEQ ID NO: 8 has higher editing activity compared to the editing activity of protein effector polypeptide comprising the SEQ ID NO: 8 when the target sequence comprises a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM, ATRRM, ATRMA, ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR (where R is purine (e.g., A or G) and M is A) at its 3' end.

[0288] In some embodiments, the protein effector polypeptides described herein are smaller compared to previously identified Cas proteins. Without wishing to be bound by a theory, the protein effector polypeptides and related systems described herein can allow an increased access to the site of target polynucleotide binding, which has several advantages. For example, they can allow easier access to the target polynucleotide for functional domains fused to the protein effector polypeptides or provided in trans. In some embodiments, the RNA:DNA duplex formed by guide molecules that form complex with the protein effector polypeptides is substantially more exposed to the environment and / or functional domains present in proximity of the DNA:RNA complex than the duplexes formed by Cas proteins known in the art. In some embodiments, the protein effector polypeptides confer a different degree of stability of the RNA:DNA duplex. In some embodiments, the protein effector polypeptides enable direct targeting of the DNA:RNA complex by one or more functional domain.

[0289] In some embodiments, the protein effector polypeptides and related systems and compositions described herein have no or limited target specificity. For example, a target polynucleotide does not need to have a specific sequence to be targeted by the protein effector polypeptides and related systems and compositions described herein. In some embodiments, the protein effector polypeptides and related systems and compositions described herein do not have a PAM requirement, in that there is no sequence requirement outside of the target sequence which defines target specificity. In some cases, the target specificity of the protein effector polypeptides and related systems and compositions described herein can be determined by the sequence of the guide molecule only, not any sequence within the target polynucleotide. In alternative embodiments, the protein effector polypeptides and related systems and compositions described herein have a target specificity, more particularly the binding of the protein effector polypeptides-guide complex is PAM-dependent. The protein effector polypeptides and related systems and compositions described herein can be modified to include PAM specificity (as described in Kleinstiver et al. 2015; Hirano et al. Mol. Cell 2016).

[0290] The protein effector polypeptide and the guide nucleic acid form a complex, also referred to as a surveillance complex. Without wishing to be bound by a theory, the surveillance complex becomes activated upon binding to the target nucleic acid. In some embodiments, the activated complex exhibits “multiple turnover” activity, whereby upon acting on (e.g., cleaving) the target nucleic acid, the activated complex remains in an activated state. In some embodiments, the activated complexexhibits “single turnover” activity, whereby upon acting on the target nucleic acid, the surveillance complex reverts to an inactive state. In some embodiments, the activated complex exhibits non-specific (i.e., “collateral”) cleavage activity, whereby the activated complex cleaves nucleic acids with no sequence similarity to the target (“collateral nucleic acids”). In some embodiments, the collateral nucleic acid substrate is a DNA (e.g., a single-stranded or a double -stranded DNA). In some embodiments, the collateral nucleic acid substrate is an RNA (e.g., a single -stranded or a double- stranded RNA).

[0291] While not bound by theory, it is believed that the protein effector polypeptides described herein can act as independent Cas effector and recognize a processed CRISPR array. Alternatively, the protein effector polypeptides described herein interact directly with a Cas enzyme, e.g., Cas 12a and bind to a target nucleic acid.

[0292] In some embodiments, the protein effector polypeptides described herein stabilize foreign RNA. In some embodiments, the protein effector polypeptides described herein comprise contain tetratricopeptide repeats (TPRs) and a serine-threonine kinase domain. It is believed that the TPRs mediate specific protein-protein interactions with Cas enzyme (e.g., Casl3a.) and Cas enzyme (e.g., Cas 13a) recognition of target RNA induces a conformational shift in Cas 13a and the kinase domain of the bound protein effector polypeptide. The conformational shift activates the kinase activity leading to phosphorylation of protein substrates and / or autophosphorylation of the kinase. Thus, protein effector polypeptide herein can be leveraged to couple specific RNA detection with phosphorylation of proteins.Nuclear localization sequences

[0293] In some embodiments of any one of the aspects described herein, the protein effector polypeptide further comprises a nuclear localization sequence (NLS) sequence or a variant thereof. The NLS can be proximal to the N- or C-terminus of the protein effector polypeptide. The NLS can be linked (e.g., fused) to the N-terminus and / or C-terminus of the protein effector polypeptide, and can be fused singly (i.e., a single NLS) or concatenated e.g., a chain of 2, 3, 4, or more NLS). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies.

[0294] In some embodiments, the protein effector polypeptide is fused to one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the protein effector polypeptide comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus) . Some exemplary NLS include, but are not limited to, those shown in Table C.Table C: Exemplary NLS sequences

[0295] In some embodiments, the NLS comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any one of the NLS sequences show in in Table C, or a homolog or ortholog thereof.Nuclear export signal sequences

[0296] In some embodiments of any one of the aspects described herein, the protein effector polypeptide further comprises a nuclear export signal (NES) sequence or a variant thereof. The NES can be proximal to the N- or C-terminus of the protein effector polypeptide. The NES can be linked (e.g., fused) to the N-terminus and / or C-terminus of the protein effector polypeptide, and can be fused singly (i.e., a single NES) or concatenated e.g., a chain of 2, 3, 4, or more NLS). When more than one NES is present, each may be selected independently of the others, such that a single NES may be present in more than one copy and / or in combination with one or more other NESs present in one or more copies.

[0297] In some embodiments, the protein effector polypeptide is fused to one or more NESs, such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NESs. In some embodiments, the protein effector polypeptide comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NESs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NESs at or near the carboxy-terminus, or a combination of these (e.g. zero or at least one or more NES at the amino- terminus and zero or at one or more NES at the carboxy terminus).

[0298] In some embodiments, a C-terminal and / or N-terminal NLS or NES is attached to the protein effector polypeptide for optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells.RuvC domain

[0299] Embodiments of the various aspects described herein include a RuvC domain. As used herein, the term “RuvC domain” refers to a conserved domain or motif of amino acids having nuclease (e.g., endonuclease) activity. A RuvC domain or segments thereof can generally be identified by alignment to known domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on known domain sequences. Generally, the RuvC domain is comprises of three discontiguous segments or subdomains, RuvC I, RuvC II, and RuvC III. The subdomains can be separated by interval sequences on the amino acid sequence of the polypeptide described herein.

[0300] The RuvC-I subdomain can share a structural similarity and / or sequence similarity to a RuvC-I domain described in the art. For example, the RuvC-I subdomain can share a structural similarity and / or sequence similarity to a RuvC-I of Cas9. In some embodiments, the RuvC-I subdomain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a homolog or ortholog of RuvC-I of Cas9.

[0301] The RuvC-II subdomain can share a structural similarity and / or sequence similarity to a RuvC-II domain described in the art. For example, the RuvC-II subdomain can share a structural similarity and / or sequence similarity to a RuvC-II of Cas9. In some embodiments, the RuvC-II subdomain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a homolog or ortholog of RuvC-II of Cas9.

[0302] The RuvC-III subdomain can share a structural similarity and / or sequence similarity to a RuvC-III domain described in the art. For example, the RuvC-III subdomain can share a structuralsimilarity and / or sequence similarity to a RuvC-III of Cas9. In some embodiments, the RuvC-III subdomain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a homolog or ortholog of a RuvC-III of Cas9.

[0303] In some embodiments of any one of the aspects described herein, the RuvC domain is a split RuvC domain. As used herein, a split RuvC domain refers to a RuvC domain having two or more RuvC motifs or subdomains, at sequentially disparate sites within a sequence, that interact in a tertiary structure to form a RuvC domain. Alternatively, the two or more RuvC motifs / subdomains can be present on different polypeptide chains and interact in a tertiary structure to form a RuvC domain.

[0304] It is noted that subdomains of the RuvC domain can be from the same species or different species. In some embodiments, the RuvC-I, RuvC-II, and RuvC-III subdomains are from different species. For example, a RuvC domain described herein can comprise a RuvC-I subdomain from a first species, a RuvC-II subdomain from a second species, and a RuvC-II subdomain from a third species. In some other embodiments, two of the RuvC-I, RuvC-II, and RuvC-III subdomains are from the same species and the other is from a different species. For example, a RuvC domain described herein can comprise RuvC -I and RuvC-II subdomains from a first species, a RuvC-III subdomain from a second species; or RuvC-I and RuvC-III subdomains from a first species, a RuvC-II subdomain from a second species; or RuvC-II and RuvC-III subdomains from a first species, a RuvC-I subdomain from a second species.

[0305] In some embodiments, the RuvC-I, RuvC-II, and RuvC-III subdomains are from the same species.

[0306] The RuvC domain can share a structural similarity and / or sequence similarity to a RuvC domain described in the art. For example, the RuvC domain can share a structural similarity and / or sequence similarity to a RuvC domain of Cas9. In some embodiments, the RuvC domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a homolog or ortholog of a RuvC domain of Cas9.

[0307] Some exemplary amino acid sequences for RuvC, RuvC -I, RuvC-II, and RuvC-III domains are described, for example, in WO2022087494, content of which is incorporated herein by reference in its entirety.HNH domain

[0308] Embodiments of the various aspects described herein include a HNH domain. As used herein, the term “HNH domain” generally refers to an endonuclease domain having characteristic histidine and asparagine residues. An HNH domain can generally be identified by alignment to known domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on known domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

[0309] The HNH domain can share a structural similarity and / or sequence similarity to a HNH domain described in the art. For example, the HNH domain can share a structural similarity and / or sequence similarity to a HNH domain of Cas9.

[0310] In some embodiments, the HNH domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a homolog or ortholog of a HNH domain of Cas9.

[0311] In some embodiments, the protein effector polypeptide comprises a RuvC domain (e.g., a split RuvC domain) and a HNH domain. For example, the protein effector polypeptide comprises both a HNH and a split RuvC domain. The HNH domain can be located within the RuvC domain. Alternatively, the HNH domain can be located within the RuvC domain. For example, the HNH domain is located between the Ruv-C II and RuvC-III subdomains of the RuvC domain.

[0312] In some embodiments, the protein effector polypeptide comprises a RuvC domain (e.g., a split RuvC domain) but no HNH domain. In some other embodiments, the protein effector polypeptide comprises a HNH domain but no RuvC domain.

[0313] Some exemplary amino acid sequences for HNH domain are described, for example, in WO2022087494, content of which is incorporated herein by reference in its entirety.Bridge helix

[0314] In some embodiments of any one of the aspects described herein, the RuvC domain is split by the insertion of a bridge helix, a HNH domain, or both. For example, the RuvC domain is split by insertion of a bridge helix. It is noted that the bridge helix can be inserted between the RuvC -I and RuvC-II subdomains, and / or between the RuvC-II and RuvC-III. In some preferred embodiments, the bridge helix is inserted between the RuvC-I and RuvC-II subdomains.

[0315] The bridge helix can share a structural similarity and / or sequence similarity to a bridge helix described in the art. For example, the bridge helix can share a structural similarity and / or sequence similarity to a bridge helix of Cas9. In some embodiments, the bridge helix comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a homolog or ortholog of a bridge helix sequence of Cas9.PLMP domain

[0316] In some embodiments of any one of the aspects described herein, the protein effector polypeptide can further comprise a conserved domain (also referred to herein as a PLMP domain), which is not present in Cas9 proteins. The PLMP domain can be located at or near the N terminus of the protein effector polypeptide.

[0317] Generally, the PLMP domain is no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, or no more than 100 amino acids in length. For example, the PLMP domain can be no more than 70 amino acids in length. For example, the PLMP domain is 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 amino acids in length. When present, the PLMP domain can be upstream of the RuvC domain (e.g., RuvC-I subdomain). For example, the PLMP domain is located within 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 amino acids upstream ofthe RuvC domain (e.g., RuvC-I subdomain).

[0318] Some exemplary amino acid sequences for the PLMP domain are described, for example, in WO2022087494, content of which is incorporated herein by reference in its entirety.Y domain

[0319] In some embodiments of any one of the aspects described herein, the protein effector polypeptide can further comprise a C-terminal domain (also referred to herein as a Y domain herein) located at or near the C-terminus of the protein effector polypeptide.

[0320] Generally, the Y domain is no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, or no more than 100 amino acids in length. For example, the Y domain can be no more than 70 amino acids in length. For example, the Y domain is 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 amino acids in length.

[0321] When present, the Y domain can be downstream of the RuvC domain (e.g., RuvC-III subdomain). For example, the Y domain can be located within 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 amino acids downstream of the RuvC domain (e.g., RuvC-III subdomain).

[0322] Some exemplary amino acid sequences for the PLMP domain are described, for example, in WO2022087494, content of which is incorporated herein by reference in its entirety.

[0323] In some embodiments of any one of the aspects described herein, the protein effector polypeptide comprises a C-terminal domain that is structurally homologous to a tudor domain. See, for example, Ren et al., Cell Res. (2014) 24: 1146- 1149. Tudor domains typically comprise a barrel- shaped beta strand fold and range in size around 50 and 60 amino acids. See, for example, Kawale, A. A. & Burmann, B.M. Inherent backbone dynamics fine-tune the functional plasticity of Tudor domains. Structure (2021), incorporated herein by reference; see, in particular, Figure 1 of Kawale showing exemplary tudor domain structure.Recognition (REC) domain

[0324] In some embodiments of any one of the aspects described herein, the protein effector polypeptide further comprises a recognition (REC) domain. The REC domain can linked / fused to the N-terminal or the C-terminal of the protein effector polypeptide. Accordingly, in some embodiments, the REC domain is linked / fused to the N-terminal protein effector polypeptide. In some other embodiments, the REC domain is linked / fused to the C-terminal protein effector polypeptide. In some embodiments, the REC domain is at an internal position of the effector polypeptide.

[0325] The REC domain can share a structural similarity and / or sequence similarity to a REC domain described in the art. For example, the REC domain can share a structural similarity and / or sequence similarity to REC domain of Cas9 or a homolog or ortholog thereof. In some embodiments, the REC domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with a homolog or ortholog of a REC of Cas9 or a homolog or ortholog thereof.

[0326] In some embodiments, the REC domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the sequence:ILTKCKVCGKNTPLRRNVRELLLENIVRFLPLESELKETLKRTILEGQQGNINKLFRKL KFNQKDWPGKNLTDIAKNKLPGRLPFCKEHFAENEKFTTIEKSTFR (SEQ ID NO: 9).

[0327] In some embodiments, the REC domain comprises an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or100% identity a REC domain of any one of SEQ ID NOs: 1-198, 221-459, 463-612, or 617-668 described in WO2021202568, content of which is incorporated herein by reference.

[0328] In some embodiments, the REC domain can be linked / fused to the N-terminal or the C- terminal of the protein effector polypeptide via a linker comprising one or more amino acids, i.e., a peptide linker.Functional mutations of protein effector polypeptides

[0329] Various mutations or modifications can be introduced into protein effector polypeptides as described herein to improve specificity and / or robustness. In some embodiments, the amino acid residues that recognize the Target Adjacent Motif (TAM) are identified. The protein effector polypeptides described herein can be modified further to recognize different TAMs, e.g., by substituting the amino acid residues that recognize PAM with other amino acid residues.

[0330] In some embodiments, the amino acid sequence of the protein effector polypeptide is mutated at one or more amino acid residues to alter its ability to functionally associate with a guide nucleic acid. In some embodiments, the protein effector polypeptide is mutated at one or more amino acid residues to alter its ability to functionally associate with a target nucleic acid. In some embodiments, the protein effector polypeptide described herein are capable of binding to or modifying a target nucleic acid molecule. In some embodiments, the protein effector polypeptide modifies both strands of the target nucleic acid molecule. However, in some embodiments, the protein effector polypeptide is mutated at one or more amino acid residues to alter its nucleic acid manipulation activity. For example, in some embodiments, the protein effector polypeptide can comprise one or more mutations which render the protein effector polypeptide incapable of cleaving a target nucleic acid. In other embodiments, the protein effector polypeptide can comprise one or more mutations such that the protein effector polypeptide is capable of cleaving a single strand of the target nucleic acid (i.e., nickase activity). In some embodiments, the protein effector polypeptide is capable of cleaving the strand of the target nucleic acid that is complementary to the strand to which the guide nucleic acid hybridizes. In some embodiments, the protein effector polypeptide is capable of cleaving the strand of the target nucleic acid to which the guide nucleic acid hybridizes.

[0331] In some embodiments, a protein effector polypeptide described herein may be engineered to comprise a deletion in one or more amino acid residues to reduce the size while retaining one or more desired functional activities (e.g., nuclease activity and the ability to interact functionally with a guide nucleic acid). The truncated protein effector polypeptide may be advantageously used in combination with delivery systems having load limitations.Functional domains

[0332] The protein effector polypeptides described herein have nuclease activity. However, they can be modified to have reduced nuclease activity, e.g., nuclease inactivation of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild-type sequence. The nuclease activity can be reduced by, for example, introducing mutations (such as amino acid insertions, deletions, or substitutions) into the nuclease domains of the protein effector polypeptides described herein. In some embodiments, catalytic residues for the nuclease activities are identified, and these amino acid residues can be substituted by different amino acid residues (e.g., glycine or alanine) to diminish the nuclease activity.

[0333] The inactivated protein effector polypeptides can comprise (e.g., via fusion protein, linker peptides, Gly4Ser (GS) peptide linkers, etc.) or be associated (e.g., via co-expression of multiple proteins) with one or more functional domains. These functional domains can have various activities, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and switch activity (e.g., light inducible). Accordingly, in some embodiments of any one of the aspects described herein, the protein effector polypeptide can be associated with a functional domain (also referred to as an effector domain herein), e.g., a domain having transposase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, nucleic acid binding activity, and / or detectable activity. For example, the protein effector polypeptide further comprises a functional domain having transposase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, nucleic acid binding activity, and / or detectable activity.

[0334] In some embodiments, the functional domain is a ligase or a functional fragment thereof. In some embodiments, the functional domain is a deaminase or a functional fragment thereof. In some other embodiments, the functional domain is a transposase or a functional fragment thereof. In yet some other embodiments, the functional domain is a reverse transcriptase or a functional fragment thereof. In some embodiments, the functional domain is a transcriptional activation domain or a functional fragment thereof. For example, the functional domain is VP64, VP16, p65, MyoDl, HSF1, RTA, SET7 / 9, a histone acetyltransferase, or a functional fragment thereof. In some embodiments, the functional domain is a transcription repression domain or a functional fragment thereof. For example,the functional domain is Kriippel associated box (KRAB), SID, or concatemers of SID (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modifying domain or a functional fragment thereof. In some embodiments, the functional domain can be an activation domain or a functional fragment thereof, such as the P65 activation domain.

[0335] In some embodiments, the functional domains are selected from the group consisting of Krilppel associated box (KRAB), VP64, VP16, Fokl, P65, HSF1, MyoDl, biotin-APEX, and functional fragments of any one of them.

[0336] The functional domain can be operably coupled to the protein effector polypeptide. One or more functional domains can be tethered or linked via a suitable linker (including, but not limited to, GlySer linkers) to the protein effector polypeptide. When there is more than one functional domain, the functional domains can be same or different. In some embodiments, all the functional domains are the same. In some embodiments, all of the functional domains are different from each other. In embodiments, at least two of the functional domains are different from each other. In some embodiments, at least two of the functional domains are the same as each other.

[0337] Generally, each functional domain is in a spatial orientation allowing for the functional domain to function in its attributed function. In other words, the positioning of the one or more functional domains on the inactivated protein effector polypeptides described herein allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect. For example, if the functional domain is a transcription activator (e.g., VP 16, VP64, or p65), the transcription activator is placed in a spatial orientation that allows it to affect the transcription of the target. Likewise, a transcription repressor (e.g., KRAB) is positioned to affect the transcription of the target, and a nuclease (e.g., Fokl) is positioned to cleave or partially cleave the target. In some embodiments, the functional domain is positioned at the N-terminus of the protein effector polypeptides described herein. In some embodiments, the functional domain is positioned at the C-terminus of the protein effector polypeptides described herein. In some embodiments, the inactivated protein effector polypeptides described herein is modified to comprise a first functional domain at the N-terminus and a second functional domain at the C-terminus.Fusion protein

[0338] In another aspect provided herein is a fusion protein comprising a protein effector polypeptide described herein covalently linked to a functional domain. For example, the fusion protein comprises a protein effector polypeptide described herein covalently linked to a functional domain having transposase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity,single-strand DNA cleavage activity, double-strand DNA cleavage activity, nucleic acid binding activity, and / or detectable activity. It is noted that the functional domain can be linked / fused to the N- terminal protein effector polypeptide. In some other embodiments, the functional domain is linked / fused to the C-terminal protein effector polypeptide. In some embodiments, the functional domain can be linked / fused to the N-terminal or the C-terminal of the protein effector polypeptide via a linker comprising one or more amino acids, i.e., a peptidyl linker.

[0339] In some embodiments, the fusion protein comprises a functional domain that is a nucleic acid editing domain.

[0340] In some embodiments, the fusion protein comprises a functional domain that is a reverse transcriptase or a functional fragment thereof.

[0341] In some embodiments, the fusion protein comprises a functional domain that is a deaminase domain or a functional fragment thereof. The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism, that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism.

[0342] In some embodiments, the deaminase or deaminase domain is a cytidine deaminase. A cytidine deaminase domain may also be referred to interchangeably as a cytosine deaminase domain. In some embodiments, the cytidine deaminase catalyzes the hydrolytic deamination of cytidine (C) or deoxycytidine (dC) to uridine (U) or deoxyuridine (dU), respectively. In some embodiments, the cytidine deaminase domain catalyzes the hydrolytic deamination of cytosine (C) to uracil (U). In some embodiments, the cytidine deaminase catalyzes the hydrolytic deamination of cytidine or cytosine in deoxyribonucleic acid (DNA). Without wishing to be bound by any particular theory, fusion proteins comprising a cytidine deaminase are useful inter alia for targeted editing, referred to herein as “base editing,” of nucleic acid sequences in vitro and in vivo.

[0343] In some embodiments, the cytidine deaminase or cytidine deaminase domain is a naturally- occurring cytidine deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the cytidine deaminase or cytidine deaminase domain is a variant of a naturally-occurring cytidine deaminase from an organism that does not occur in nature. For example, in some embodiments, the cytidine deaminase or cytidine deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical toa naturally-occurring cytidine deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse

[0344] In some embodiments, the cytidine deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the cytidine deaminase is an APOBEC1 deaminase. In some embodiments, the cytidine deaminase is an APOBEC2 deaminase. In some embodiments, the cytidine deaminase is an APOBEC3 deaminase. In some embodiments, the cytidine deaminase is an APOBEC3A deaminase. In some embodiments, the cytidine deaminase is an APOBEC3B deaminase. In some embodiments, the cytidine deaminase is an APOBEC3C deaminase. In some embodiments, the cytidine deaminase is an APOBEC3D deaminase. In some embodiments, the cytidine deaminase is an APOBEC3E deaminase. In some embodiments, the cytidine deaminase is an APOBEC3F deaminase. In some embodiments, the cytidine deaminase is an APOBEC3G deaminase. In some embodiments, the cytidine deaminase is an APOBEC3H deaminase. In some embodiments, the cytidine deaminase is an APOBEC4 deaminase. In some embodiments, the cytidine deaminase is an activation-induced deaminase (AID). In some embodiments, the cytidine deaminase is a vertebrate cytidine deaminase. In some embodiments, the cytidine deaminase is an invertebrate cytidine deaminase. In some embodiments, the cytidine deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the cytidine deaminase is a human cytidine deaminase. In some embodiments, the cytidine deaminase is a rat cytidine deaminase, e.g., rAPOBECl. In some embodiments, the cytidine deaminase is a Petromyzon marinus cytidine deaminase 1 (pmCDAl). In some embodiments, the cytidine deaminase is a human APOBEC3G. In some embodiments, the cytidine deaminase is a fragment of the human APOBEC3G. In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R and D317R mutation. In some embodiments, the deaminase is a fragment of the human APOBEC3G and comprising mutations corresponding to the D316R and D317R mutations.

[0345] In some embodiments, the cytidine deaminase domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the cytidine deaminase domain of any one of the exemplary cytidines deaminase sequences shown in Table DI. In some embodiments, the cytidine deaminase domain comprises the amino acid sequence of any one of the exemplary cytidines deaminase sequences shown in Table DI. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).Table DI: Exemplary cytidine deaminase sequences

[0346] In another example, the fusion protein comprises a functional domain that is an adenosine deaminase domain or a functional fragment thereof. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, catalyzing the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism, such as a bacterium. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium, such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may be missing one or more N- terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA maybe missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N- terminal methionine.

[0347] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any one of the exemplary adenosine deaminase sequences shown in Table D2, or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 62-84, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in in any one of the exemplary adenosine deaminase sequences shown in Table D2, or any of the adenosine deaminases provided herein.Table D2: Exemplary adenosine deaminase sequences

[0348] In some cases, the adenosine deaminase is double-stranded RNA-specific adenosine deaminase (ADAR). Examples of ADARs include those described Yiannis A Savva et al., The ADAR protein family, Genome Biol. 2012; 13(12): 252, which is incorporated by reference in its entirety. In some examples, the ADAR may be hADARl. In certain examples, the ADAR may be hADAR2. The sequence of hADAR2 may be that described under Accession No. AF525422.1.

[0349] In some cases, the deaminase may be a deaminase domain, e.g., a deaminase domain of ADAR (“ADAR-D”). In one example, the deaminase may be the deaminase domain of hADAR2 (“hADAR2-D), e.g., as described in Phelps KJ et al., Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2. Nucleic Acids Res. 2015 Jan;43(2): 1123-32, which is incorporated by reference herein in its entirety. In a particular example, the hADAR2-D has a sequence comprising amino acid 299-701 of hADAR2-D, e.g., amino acid 299-701 of the sequence under Accession No. AF525422.1. In certain examples, the adenosine deaminase may comprise one or more of the mutations: E488Q based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In oneembodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, based on amino acid sequence positions of hADAR2- D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N based on amino acidsequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T based on amino acid sequence positions of hADAR2-D, and mutations in a homologous ADAR protein corresponding to the above. In some examples, provided herein includes a mutated adenosine deaminase e.g., an adenosine deaminase comprising one or more mutations of E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T. In some examples, provided herein includes a mutated adenosine deaminase e.g., an adenosine deaminase comprising E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, and S661T. In some examples, provided herein includes a mutated adenosine deaminase e.g., an adenosine deaminase comprising E488Q, V351G, S486A, T375S, S370C, P462A, N597I, L332I, I398V, K350I, M383L, D619G, S582T, V440I, S495N, K418E, S661T, and S375N. In one embodiment, the adenosine deaminase may be a tRNA-specific adenosine deaminase or a variant thereof. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: W23L, W23R, R26G, H36L, N37S, P48S, P48T, P48A, I49V, R51L, N72D, L84F, S97C, A106V, D108N, H123Y, G125A, A142N, S146C, D147Y, R152H, R152P, E155V, I156F, K157N, K161T, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more ofthe mutations: D108N based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: Al 06V, D108N, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more ofthe mutations: A106V, D108N, D147Y, E155V, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, A142N, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to theabove. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A 106V, D108N, D147Y, El 55V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, El 55 V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, El 55V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, A142N, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R5 IL, S146C, K157N, P48S, W23R, P48A, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, W23R, P48A, A142N, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, W23R, P48A, R152P, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above. In one embodiment, the adenosine deaminase may comprise one or more of the mutations: A106V, D108N, D147Y, E155V, L84F, H123Y, I156F, H36L, R51L, S146C, K157N, P48S, W23R, P48A, R152P, A142N, based on amino acid sequence positions of E. coli TadA, and mutations in a homologous deaminase protein corresponding to the above.

[0350] In some embodiments, the fusion protein further comprises a second adenosine deaminase domain. The second adenosine deaminase can be an ecTadA domain, a variant, or a functional fragment thereof. In some embodiments, the first and second adenosine deaminase domain independently comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity (e.g., when translated into a polypeptide) to any one of to any one of the sequences in Table D2. In one example, both the first and second adenosine deaminase domains are an ecTadA domain, a variant, or a functional fragment thereof.

[0351] In some embodiments, the fusion protein comprises a functional domain that is uracil glycosylase inhibitor (UGI) domain or a functional fragment thereof.

[0352] The term “uracil glycosylase inhibitor” or “UGI,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, aUGI domain comprises a wild-type UGI or a UGI as set forth in Table E. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in Table E. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in TABLE E. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in TABLE E, or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in TABLE E. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as “UGI variants.” A UGI variant shares homology to UGI, or a fragment thereof. For example, a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in Table E. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in Table E.Table E: Exemplary UGI sequences

[0353] In yet another example, the fusion protein comprises a functional domain that is reverse transcriptase domain or a functional fragment thereof. A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by retroviruses to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes, and by some non-retroviruses such as the hepatitis B virus, a member of the Hepadnaviridae, which are dsDNA-RT viruses. Retroviral RT has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H, and DNA- dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single -stranded RNA into double -stranded cDNA. In an embodiment, the RT domain of a reverse transcriptase is used in the present invention. The domain may include only the RNA-dependent DNA polymerase activity. In some examples, the RT domain is non- mutagenic, i.e., does not cause mutation in the donor polynucleotide (e.g., during the reverse transcriptase process). In some examples, the RT domain may be non-retron RT, e.g., a viral RT or a human endogenous RTs. In some examples, the RT domain may be retron RT or DGRs RT. In some example, the RT may be less mutagenic than a counterpart wildtype RT. In one embodiment, the RT herein is not mutagenic. Exemplary the reverse transcriptase include, but are not limited to, Human immunodeficiency virus (HIV) RT, Avian myeloblastosis virus (AMV) RT, Moloney murine leukemia virus (M-MLV) RT a group II intron RT, a group II intron-like RT, or a chimeric RT. In some embodiments, the functional domain comprises modified forms of these RTs, such as, engineered variants of Avian myeloblastosis virus (AMV) RT, Moloney murine leukemia virus (M-MLV) RT, or Human immunodeficiency virus (HIV) RT (see, e.g., Anzalone, et al., Search-and-replace genome editing without double-strand breaks or donor DNA, Nature. 2019 Dec;576(7785): 149-157). Without wishing to be bound by any particular theory, fusion proteins comprising a reverse transcriptase domain are useful inter alia for targeted editing, referred to herein as “prime editing,” of nucleic acid sequences in vitro and in vivo.

[0354] Also provided herein is a complex comprising a fusion protein described herein bound to a guide nucleic acid (e.g., gRNA or coRNA). In another aspect, provided herein is a complex comprisinga coRNA described herein bound to a transposon family encoded nuclease or a homolog or ortholog thereof. For example, the complex comprises coRNA described herein bound to a IS200 / IS605 transposon family encoded nuclease (such as IscB, Isrb, and TnpB) or a homolog or ortholog thereof. Complexes comprising a guide nucleic acid (e.g., gRNA or coRNA) bound, e.g., to protein effector polypeptide or a fusion protein described herein, or to a transposon family encoded nuclease or a homolog or ortholog thereof are also referred to as engineered, non-natural DNA and RNA targeting complexes, DNA and RNA targeting systems, and DNA and RNA targeting compositions herein.

[0355] In some embodiments, a DNA and RNA targeting complex described herein produces fewer indels in a target sequence that does not comprise the canonical TAM (5’-ATAAA) at is 3 ’-end as compared to the number of indels produced by a complex comprising a wild-type IscB and wild-type gRNA. For example, a DNA and RNA targeting complex described herein produces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold fewer indels in atarget sequence that does not comprise the canonical TAM (5’-ATAAA) at is 3 ’-end as compared to the number of indels produced by a complex comprising a wild-type IscB and wild-type gRNA. In some embodiments, indels can be measured using high-throughput sequencing.

[0356] In some embodiments, a DNA and RNA targeting complex described herein, where the protein effector polypeptide comprises a deaminase domain, exhibits an increased deamination efficacy in a target sequence that does not comprise the canonical TAM (5’-ATAAA) at is 3 ’-end as compared to the deamination activity of a complex comprising a wild-type IscB and wild-type gRNA. For example, the deamination efficiency of a DNA and RNA targeting complex, where the protein effector polypeptide comprises a deaminase domain, in a target sequence having a 3 ’-end that is not directly adjacent to the sequence 5’-ATAAA is at least is at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold higher as compared to the deamination efficiency of a complex comprising a wild- type IscB and wild-type gRNA. The deamination activity can be measured using a deamination assay, PCR, sequencing or any combination thereof.

[0357] In some embodiments, a DNA and RNA targeting complex described herein exhibits a lower or decreased off-target activity, i.e., modification of a non-target sequence, as compared to the off-target activity of a complex comprising a wild-type IscB and wild-type gRNA. For example, the off-target activity of a DNA and RNA targeting complex described herein, is at least at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold lower than the off-target activity of acomplex comprising a wild-type IscB and wild-type gRNA.Guide nucleic acids (coRNAs)

[0001] As used herein, a “guide nucleic acid”, “gNA” refers to a nucleic acid that facilitates the targeting of protein effector polypeptide described herein to a target nucleic acid. The guide nucleic acid are also referred to as coRNA or guide RNA (“gRNA”) herein. A guide nucleic acid can be RNA, DNA or a mix of RNA / DNA.

[0002] The ability of a guide nucleic acid to direct sequence-specific binding of a protein effector polypeptide described herein a target nucleic acid sequence can be assessed by any suitable assay. For example, components sufficient to form a complex comprising a protein effector polypeptide and the guide nucleic acid to be tested, can be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay described in WO2022087494, content of which is incorporated herein by reference. Similarly, cleavage of a target nucleic acid sequence (or a sequence in the vicinity thereof) can be evaluated in a test tube by providing the target nucleic acid sequence, and components sufficient to form a complex comprising a protein effector polypeptide and the guide nucleic acid to be tested, and comparing binding or rate of cleavage at or in the vicinity of the target sequence between the test and control guide nucleic acid reactions. Other assays are possible, and will occur to those skilled in the art.

[0003] Some exemplary gRNAs amenable to various aspects described herein are described, for example, in PCT publications WO2022087494, W02020180699, WO2021226363, WO2022261292, WO2021119275, WO2019237069, WO2018107028, WO2019067872 and WO2019067992, content of each of which is incorporated herein by reference in their entireties. coRNA

[0004] Generally, a coRNA or guide RNA comprises a conserved region also referred to as a conserved portion. Without wishing the be bound a theory, the coRNA interacts, e.g., associates or binds with a protein effector polypeptide via the conserved region. In some embodiments, the conserved region can comprise one or more hairpin or stem-loop structures. The hairpin closest to the 5 ’-end of the conserved region can be referred to as a 5 ’-stem -loop, 5 ’-hairpin or 5 ’-proximal hairpin. The hairpin closest to the 3 ’-end of the conserved region can be referred to as a 3 ’-stem-loop, 3 ’-hairpin or 3’- proximal hairpin. When the conserved region comprises three or more hairpins, the hairpins that are not near 5’ - or 3’- end of the conserved region, (i.e., not a 5 ’-hairpin or not a 3 ’-hairpin) can be referred to as internal hairpins, internal stem-loop, non-end hairpins or non-end stem-loops herein. In some embodiments, the conserved region has a secondary structure comprising three hairpins. Withoutwishing to be bound by a theory, in some embodiments, the conserved region has a secondary structure comprising three hairpins as shown in FIGS. 3B and 4A-4C.

[0005] In some embodiments, a coRNA described herein comprises a conserved portion of a coRNA, e.g., a wild-type coRNA (parent coRNA), where the conserved region of the coRNA comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more) modifications as compared to the conserved region of the parent or wild-type coRNA. In some embodiments, one or more nucleotides from the conserved region of the parent or wild-type coRNA are not present in the coRNA described herein.5 ’-hairpin

[0006] In various embodiments, the conserved region of coRNA comprises a hairpin or stem-loop structure where at least a position of a hairpin or stem-loop structure (e.g., the first base-paired nucleotide at the 5’-end of the stem of the hairpin) is within 45 nucleotides, counting from the 5’-end of the conserved region. For example, the conserved region of the coRNA comprises a hairpin or stem- loop structure the first base-paired nucleotide at the 5 ’ -end of the stem of the hairpin is within 40, within35, within 30, within 25, within 20, within 15, within 10 or within 5 nucleotides from the 5 ’-end of the conserved region. In some embodiments, the first base-pair nucleotide at the 5 ’-end of the stem of the hairpin is within 15, within 14, within 13, within 12, within 11, within 10, within 9, within 8, within 8, within 7, within 6, within 5, within 5, within 4, within 3, within 2 nucleotides from the 5 ’-end of the conserved region.

[0007] In some embodiments, the 5 ’-hairpin comprises a modification compared to the conserved region of the parent conserved region. For example, one or more nucleotides from the 5 ’-end hairpin of the parent or wild-type conserved region are removed or deleted. In some embodiments, 3 or more contiguous or non-contiguous nucleotides from the 5 ’-end hairpin of the parent or wild-type conserved region are removed or deleted in the conserved region of a coRNA described herein. For example, from about 8 to about 50 contiguous or non-contiguous nucleotides from the 5 ’-end hairpin of the parent or wild-type conserved region are removed or deleted in the conserved region of a coRNA described herein. In some embodiments, a coRNA described herein comprises a conserved region where 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 contiguous or non-contiguous nucleotides are removed or deleted from the 5 ’-hairpin compared to the wild-type or parent coRNA. For example, a coRNA described herein comprises a conserved region where 8, 12, 16, 20, 23, 27, 28, 31, 35, 39, 42, or 46 contiguous or non-contiguous nucleotides are removed or deleted from the 5 ’-hairpin compared to the wild-type or parent coRNA. In some embodiments, the coRNA comprises a conserved region where 8, 20, 35, 42, or 46 contiguous or non-contiguous nucleotides are removed or deleted from the 5 ’-hairpin compared to the wild-type or parent coRNA.

[0008] In some embodiments, a coRNA described herein comprises a conserved region where 3, 7, 11, 15, 18, 22, 26, 30, 34, 37 or 41 contiguous or non-contiguous nucleotides are removed or deleted from the 5 ’-hairpin compared to the wild-type or parent coRNA. For example, the coRNA comprises a conserved region where 3, 15, 30, 37, or 41 contiguous or non-contiguous nucleotides are removed or deleted from the 5 ’-hairpin compared to the wild-type or parent coRNA.

[0009] In some embodiments, nucleotides at position 12-57, 14-55, 16-54, 18-52, 20-50, 22-48, 24-46, 26-45, 28-43, 30-41 or 32-39 of the wild-type or parent conserved region are removed or deleted (counting from 5 ’-end of the conserved region of the parent coRNA). For example, nucleotides at position 12-57, 14-55, 18-52, 24-46, or 26-45 of the wild-type or parent conserved region are removed or deleted (counting from 5 ’-end of the conserved region of the parent coRNA). In some embodiments, nucleotides at position 11-56, 13-54, 15-53, 17-51, 19-49, 21-47, 23-45, 25-44, 27-42, 29-41 or 32-39 of the wild-type or parent conserved region are removed or deleted (counting from first base-paired nucleotide at 5’-end of the 5’-hairpin). For example, nucleotides at position 11-56, 13-54, 17-51, 23- 45, or 25-44 of the wild-type or parent conserved region are removed or deleted (counting from first base-paired nucleotide at 5 ’-end of the 5 ’-hairpin).

[0010] It is noted that the nucleotides removed or deleted from the conserved region of the wild- type or parent coRNA’s 5’-hairpin can be replaced with a linker. For example, the deleted or the removed nucleotides can be replaced with a nucleotide or a non-nucleotide linker. In some embodiments, the nucleotides removed or deleted from the conserved region of the wild-type or parent coRNA’s 5’-hairpin are replaced with a nucleotide sequence, e.g., a single-stranded nucleic acid comprising from about comprising from about 4 nucleotides to about 15 nucleotides. For example, the deleted or the removed nucleotides can be replaced with nucleotide linker comprising 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the deleted or the removed nucleotides can be replaced with a nucleotide sequence comprising 4, 5 or 6 nucleotides. In some embodiments, the deleted or the removed nucleotides are replaced with the nucleotide sequence GAAA or GAAAA. For example, the deleted or the removed nucleotides are replaced with the nucleotide sequence GAAAA

[0011] The 5’-hairpin comprises a double-stranded region (i.e., stem portion) and a single -stranded region (i.e., a pin-loop) connecting the two strands forming the stem portion of the hairpin. The stem portion of the 5 ’-hairpin can comprise a double -stranded region comprising 4 or more nucleotide base pairs. For example, the stem portion of the 5 ’-hairpin can comprise a double-stranded region comprising from about 5 to about 30 nucleotide base-pairs. In some embodiments, the stem portion of the 5 ’-hairpin comprises a double-stranded region comprising from about 8 to about 25 nucleotide base- pairs. For example, the stem portion of the 5 ’-hairpin comprises a double-stranded region comprising about 9 nucleotide base-pairs, about 10 nucleotide base-pairs, about 11 nucleotide base-pairs, about 12 nucleotide base-pairs, about 13 nucleotide base-pairs, about 14 nucleotide base-pairs, about 15 base pairs nucleotide base-pairs, about 16 base pairs nucleotide base-pairs, about 17 base pairs nucleotidebase-pairs, about 18 base pairs nucleotide base-pairs, about 19 base pairs nucleotide base-pairs, or about 20 base pairs nucleotide base-pairs. In some embodiments, the stem portion of the 5 ’-hairpin comprises a double-stranded region comprising 8, 9, 10, 11 or 12 nucleotide base pairs. In some preferred embodiments, the stem portion of the 5’-hairpin comprises a double-stranded region comprising 9, 10 or 11 nucleotide base pairs. For example, the stem portion of the 5 ’-hairpin comprises a double -stranded region comprising 10 nucleotide base pairs.

[0012] In some embodiments, the stem portion of the 5 ’-hairpin comprises a double-stranded region comprising 1, 2, 3, 4, 5 6 or 7 nucleotide base-pairs.

[0013] In some embodiments, the stem portion of the 5 ’-hairpin comprises a mismatch. For example, the stem portion of the 5’-hairpin comprises a mismatch (e.g., an A:C mismatch) at position 2, 3, 4, 5, 6, 7, 8, or 9, counting from the first base-paired nucleotide at the 5 ’-end of the 5 ’-hairpin.

[0014] In some embodiments, the stem portion of the 5 ’-hairpin comprises a double-stranded region comprising 8, 9, 10, 11, or 12 nucleotide base-pairs and the stem portion further comprises a mismatch in the stem portion. For example, the stem portion of the 5 ’-hairpin comprises a double- stranded region comprising 8, 9, 10, 11, or 12 nucleotide base-pairs and the stem portion further comprises a mismatch (e.g., an A:C mismatch) at position 3, 4, 5, 6, 7, or 8, counting from the first base-paired nucleotide at the 5 ’-end of the 5 ’-hairpin. In some embodiments, the mismatch (e.g., an A:C mismatch) is at position 3, 4, 5, 6 or 7. For example, the mismatch (e.g., an A:C mismatch) is at position 4 or 5, counting from the first base-paired nucleotide at the 5’-end of the 5’-hairpin.

[0015] The pin-loop portion of the 5 ’-hairpin can be 3 nucleotides or longer. For example, the pin-loop portion of the 5 ’-hairpin can be from about 4 nucleotides to about 15 nucleotides. For example, the pin-loop portion of the 5’-hairpin can be 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the pin-loop portion of the 5 ’-hairpin is 4, 5 or 6 nucleotides in length.

[0016] A total length of the 5 ’-hairpin, e.g., from the first base-paired nucleotide at the 5 ’-end to the last bas-paired nucleotide at the 3 ’-end of the hairpin, can be from about from about 20 nucleotides to about 65 nucleotides. For example, the 5’-hairpin can comprise from about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, or about 45 nucleotides. In some embodiments, the 5 ’-hairpin comprises 23, 24, 25, 26 or 27 nucleotides. For example, the 5’-hairpin comprises, 24, 25 or 26, e.g., 25 nucleotides.

[0017] The 5’-hairpin can comprise a nucleic acid modification, e.g., to stabilize the hairpin. For example, the 5 ’-hairpin can comprise a nucleic acid modification selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof. The modification can be present at any location in the 5’-hairpin. In some embodiments, the stem portion of the 5’- hairpin comprises a nucleic acid modification. In some embodiments, the pin-loop portion of the 5’-hairpin comprises a nucleic acid modification. In yet some other embodiments, both the stem portion and the pin-loop portion comprise independently selected nucleic acid modification.

[0018] In some embodiments, the stem portion and / or the pin-loop portion of the 5 ’-hairpin comprises a modified nucleotide selected independently from the group consisting of 2’-O-methyl (2’- OMe) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’- C-methylene (locked nucleic acid, LNA) nucleotides, 2’-O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl -carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene (Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-M0E) nucleotides, 2’-O-allyl nucleotides, 2’-C- allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-0-NMA) nucleotides, a 2'-O- dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, 2’- F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; a non-natural or modified nucleobase; or a combination thereof. For example, the stem portion and / or the pin-loop portion of the 5 ’-hairpin comprises a 2’-O-methyl modified nucleotide. In another example, the stem portion and / or the pin-loop portion of the 5 ’-hairpin comprises a 2 ’-fluoro modified nucleotide.

[0019] The 5’-hairpin can also comprise one or more (e.g., 1, 2, 3, 4, 5 or more) non-natural or modified nucleobases. The non-natural or modified nucleobase can be present in the stem portion or the pin-loop portion of the 5 ’-hairpin. In some embodiments, the stem portion of the 5 ’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) non-natural or modified nucleobases. In another embodiment, the pin-loop portion of the 5 ’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) non-natural or modified nucleobases. In yet some other embodiments, both the stem portion and the pin-loop portion of the 5’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) independently selected non-natural or modified nucleobases.

[0020] The 5’-hairpin can also comprise at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkages. Exemplary modified intemucleoside linkages are described herein and include, but are not limited to, phosphorothioate and MMI linkages. Like other modifications, a modified intemucleotide linkage can be present in the stem portion and / or the pin-loop portion of the 5 ’-hairpin.

[0021] In some embodiments, the stem portion of the 5’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In another embodiment, the pin-loop portion of the 5’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In yet some other embodiments, both the stem portion and the pin-loop portion of the 5 ’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) independently selected modified intemucleoside linkages (e.g.,phosphorothioate and / or MMI linkages). In some embodiments, the 5 ’-hairpin comprises a modified intemucleoside linkage (e.g., phosphorothioate and / or MMI linkage) between the stem portion and the pin-loop portion of the 5 ’-hairpin.

[0022] Without wishing to be bound by a theory, increasing the stability, e.g., stability of the stem of the 5’-hairpin can provide increased or higher nucleic acid modification, e.g., editing activity and / or efficiency. Accordingly, in some embodiments, the stem portion of the 5 ’-hairpin comprises a duplex stabilizing modification. Some exemplary duplex stabilizing modifications include, but are not limited to, 2’-F nucleotides, 2’-0Me nucleotides, 2 ’-methoxy ethyl nucleotides, 2,6-diaminopurine nucleotides, 5-methyl cytidine, N4-ethyl cytidine, 5-propynyl cytidine, 5-propynyl uridine, 5-hydroxybutynl-2’- deoxyuridine, 8-aza-7-deazaguanosine, a locked nucleic acid (LNA), and / or covalent cross-linking of two strands of the duplex. In some embodiments, the stem portion comprises a covalent linkage between two nucleotides, optionally the covalent linkage is at a position other than 5 ’and 3’ of the nucleotides.

[0023] In some embodiments, a duplex stabilizing modification comprises a A:U base pair in the stem of the 5 ’-hairpin to a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5, 6, 7 or more A:U base pairs in the stem of the 5’-hairpin ton G:C base-pairs. In some embodiments, a duplex stabilizing modification comprises replacing a A:U base pair in the stem of the 5 ’-hairpin with a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5, 6, 7 or more A:U base pairs in the stem of the 5’-hairpin ton G:C base-pairs. In some embodiments, a duplex stabilizing modification comprises replacing mismatch in the stem of the 5 ’-hairpin with a G:C or A:U base-pair.

[0024] It is known that guanine tetrads are formed by sequences rich in guanine, such as GGGGC. Thus, in order to reduce formation of guanine tetrads, G and C in the G:C base-pair replacing a A:U base-pair or a mismatch are positioned so that there are no more than 5, preferably no more than 4, and more preferably no more than 3 consecutive G in the sequence of the stem.

[0025] In some embodiments, an A:C mismatch in the stem of the 5 ’-hairpin of a wild-type or parent coRNA conserved region is replaced with a G:C base-pair. For example, the A in the mismatch is replaced by G, or the A is replaced by C and the Cis replaced by G. In some other embodiments, the A:C mismatch in the stem of the 5’-hairpin is replaced by an A:U base-pair. For example, the C in the mismatch is replaced by U, or the A is replaced by U and the Cis replaced by A.

[0026] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5 or more) A:U base-pairs present in the stem of the 5 ’-hairpin of the wild-type or parent coRNA conserved region are deleted.

[0027] In some embodiments, one or more nucleotides present at the 5 ’-end of the wild-type or parent coRNA conserved region, that are not involved in forming the 5 ’-hairpin are deleted.3 ’-hairpin

[0028] In various embodiments, the conserved region of coRNA comprises a hairpin or stem-loopstructure where at least a position of a hairpin or stem-loop structure (e.g., the first base-paired nucleotide at the 3’-end of the stem of the hairpin) is within 50 nucleotides, counting from the 3’-end of the conserved region. For example, the conserved region of the coRNA comprises a hairpin or stem- loop structure the first base-paired nucleotide at the 3’-end of the stem of the hairpin is within 45, within 40, within 35, within 30, within 25, within 20, within 15, within 10 or within 5 nucleotides from the 3’- end of the conserved region. In some embodiments, the first base-pair nucleotide at the 3 ’-end of the stem of the 3’-hairpin is within 35, within 34, within 33, within 32, within 31, within 30, within 29, within 28, within 27, within 26, within 25, within 24, within 23, within 22, within 21, within 20, within 19, within 18, within 17, within 16, within 15, within 14, within 13, within 12, within 11, or within 10 nucleotides from the 3 ’-end of the conserved region. For example, the first base-pair nucleotide at the 3’-end of the stem of the 3’-hairpin is within 20, within 19, within 18, within 17, within 16, within 15, within 14, within 13, within 12, within 11, or within 10 nucleotides from the 3 ’-end of the conserved region. In some embodiments, the first base-pair nucleotide at the 3 ’-end of the stem of the 3 ’-hairpin is within 9, within 8, within 7, within 6, within 5, within 4, within 3, within 2 nucleotides from the 3’- end of the conserved region.

[0029] In some embodiments, the 3 ’-hairpin comprises a modification compared to the conserved region of the parent conserved region. For example, one or more nucleotides from the 3 ’-hairpin of the parent or wild-type conserved region are removed or deleted. In some embodiments, 1 or at least 2 or more contiguous or non-contiguous nucleotides from the 3 ’-end hairpin of the parent or wild-type conserved region are removed or deleted in the conserved region of a coRNA described herein. For example, from 1 to about 25 contiguous or non-contiguous nucleotides from the 3 ’-end hairpin of the parent or wild-type conserved region are removed or deleted in the conserved region of a coRNA described herein. In some embodiments, a coRNA described herein comprises a conserved region where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous or non-contiguous nucleotides are removed or deleted from the ’-hairpin compared to the wild-type or parent coRNA. For example, a coRNA described herein comprises a conserved region where 2, 6, 8, 10, 12, 16, or 20 contiguous or non-contiguous nucleotides are removed or deleted from the 3 ’-hairpin compared to the wild-type or parent coRNA. In some embodiments, the coRNA comprises a conserved region where 2, 10, 12, or 16 contiguous or non-contiguous nucleotides are removed or deleted from the 3 ’-hairpin compared to the wild-type or parent coRNA.

[0030] In some embodiments, a coRNA described herein comprises a conserved region where 6, 10, 12, 14, 16, 20 or 22 contiguous or non-contiguous nucleotides are removed or deleted from the 3 ’-hairpin compared to the wild-type or parent coRNA. For example, the coRNA comprises a conserved region where 6, 14, 16, or 20 contiguous or non-contiguous nucleotides are removed or deleted from the 3 ’-hairpin compared to the wild-type or parent coRNA.

[0031] In some embodiments, nucleotides at position 159-172, 160-171, 161-170, 163-168, 158-173, 156-175 or 154-177 of the wild-type or parent conserved region are removed or deleted (counting from 5’-end of the conserved region of the parent coRNA). For example, nucleotides at position 159- 172, 163-168, 158-173, or 156-175 of the wild-type or parent conserved region are removed or deleted (counting from 5 ’ -end of the conserved region of the parent coRNA. In some embodiments, nucleotides at position 6-19, 7-18, 8-17, 10-15, 5-20, 3-22, or 1-24 of the wild-type or parent conserved region are removed or deleted (counting from first base-paired nucleotide at 5’-end of the 3’-hairpin). For example, nucleotides at position 6-19, 10-15, 5-20, or 3-22 of the wild-type or parent conserved region are removed or deleted (counting from first base-paired nucleotide at 5 ’-end of the 3 ’-hairpin).

[0032] It is noted that the nucleotides removed or deleted from the conserved region of the wild- type or parent coRNA’s 3’-hairpin can be replaced with a linker. For example, the deleted or the removed nucleotides can be replaced with a nucleotide or a non-nucleotide linker. In some embodiments, the nucleotides removed or deleted from the conserved region of the wild-type or parent coRNA’s 3’-hairpin are replaced with a nucleotide sequence, e.g., a single-stranded nucleic acid comprising from about comprising from about 4 nucleotides to about 15 nucleotides. For example, the deleted or the removed nucleotides can be replaced with nucleotide linker comprising 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the deleted or the removed nucleotides can be replaced with a nucleotide sequence comprising 4, 5 or 6 nucleotides. In some embodiments, the deleted or the removed nucleotides are replaced with the nucleotide sequence GAAA or GAAAA. For example, the deleted or the removed nucleotides are replaced with the nucleotide sequence GAAA

[0033] The 3’-hairpin comprises a double-stranded region (i.e., stem portion) and a single -stranded region (i.e., pin-loop portion) connecting the two strands forming the stem portion of the hairpin. The stem portion of the 3 ’-hairpin can comprise a double -stranded region comprising 4 or more nucleotide base pairs. For example, the stem portion of the 3 ’-hairpin can comprise a double-stranded region comprising from about 3 to about 20 nucleotide base-pairs. In some embodiments, the stem portion of the 3 ’-hairpin comprises a double-stranded region comprising from about 4 to about 15 nucleotide base- pairs. For example, the stem portion of the 3 ’-hairpin comprises a double-stranded region comprising about 5, about 6, about 7, about 8, about 9 nucleotide base-pairs, about 10 nucleotide base-pairs, about 11 nucleotide base-pairs, about 12 nucleotide base-pairs, about 13 nucleotide base-pairs, about 14 nucleotide base-pairs, or about 15 nucleotide base-pairs. In some embodiments, the stem portion of the 3’-hairpin comprises a double-stranded region comprising 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide base pairs. In some preferred embodiments, the stem portion of the 3 ’-hairpin comprises a double -stranded region comprising 3, 4, 5, or 6 nucleotide base pairs. For example, the stem portion of the 3 ’-hairpin comprises a double -stranded region comprising 3 or 4 nucleotide base pairs.

[0034] The pin-loop portion of the 3 ’-hairpin can be 3 nucleotides or longer. For example, the pin-loop portion of the 3 ’-hairpin can be from about 4 nucleotides to about 15 nucleotides. For example, the pin-loop portion of the 3’-hairpin can be 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Insome embodiments, the pin-loop portion of the 3 ’-hairpin is 4, 5 or 6 nucleotides in length.

[0035] A total length of the 3 ’-hairpin, e.g., from the first base-paired nucleotide at the 5 ’-end to the last base-paired nucleotide at the 3 ’-end of the hairpin, can be from about 8 nucleotides to about 25 nucleotides.

[0036] For example, the 3’-hairpin can comprise about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides. In some embodiments, the 3 ’-hairpin comprises 23, 24, 25, 26 or 27 nucleotides. For example, the 3 ’-hairpin comprises 11, 12, 13, 14 or 15 nucleotides.

[0037] The 3’-hairpin can comprise a nucleic acid modification, e.g., to stabilize the hairpin. For example, the 3 ’-hairpin can comprise a nucleic acid modification selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof. The modification can be present at any location in the 3’-hairpin. In some embodiments, the stem portion of the 3’- hairpin comprises a nucleic acid modification. In some embodiments, the pin-loop portion of the 3’- hairpin comprises a nucleic acid modification. In yet some other embodiments, both the stem portion and the pin-loop portion comprise independently selected nucleic acid modification.

[0038] In some embodiments, the stem portion and / or the pin-loop portion of the 3 ’-hairpin comprises a modified nucleotide selected independently from the group consisting of 2’-O-methyl (2’- OMe) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’- C-methylene (locked nucleic acid, LNA) nucleotides, 2’-O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl -carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene (Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-M0E) nucleotides, 2’-O-allyl nucleotides, 2’-C- allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-0-NMA) nucleotides, a 2'-O- dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, 2’- F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; a non-natural or modified nucleobase; or a combination thereof. For example, the stem portion and / or the pin-loop portion of the 3 ’-hairpin comprises a 2’-O-methyl modified nucleotide. In another example, the stem portion and / or the pin-loop portion of the 3 ’-hairpin comprises a 2 ’-fluoro modified nucleotide.

[0039] The 3’-hairpin can also comprise one or more (e.g., 1, 2, 3, 4, 5 or more) non-natural or modified nucleobases. The non-natural or modified nucleobase can be present in the stem portion or the pin-loop portion of the 3 ’-hairpin. In some embodiments, the stem portion of the 3 ’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) non-natural or modified nucleobases. In another embodiment, the pin-loop portion of the 3 ’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more)non-natural or modified nucleobases. In yet some other embodiments, both the stem portion and the pin-loop portion of the 3’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) independently selected non-natural or modified nucleobases.

[0040] The 3’-hairpin can also comprise at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkages. Exemplary modified intemucleoside linkages are described herein and include, but are not limited to, phosphorothioate and MMI linkages. Like other modifications, a modified intemucleotide linkage can be present in the stem portion and / or the pin-loop portion of the 3 ’-hairpin.

[0041] In some embodiments, the stem portion of the 3’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In another embodiment, the pin-loop portion of the 3’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In yet some other embodiments, both the stem portion and the pin-loop portion of the 3 ’-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) independently selected modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In some embodiments, the 3 ’-hairpin comprises a modified intemucleoside linkage (e.g., phosphorothioate and / or MMI linkage) between the stem portion and the pin-loop portion of the 3 ’-hairpin.

[0042] Without wishing to be bound by a theory, increasing the stability, e.g., stability of the stem of the 3’-hairpin can provide increased or higher nucleic acid modification, e.g., editing activity and / or efficiency. Accordingly, in some embodiments, the stem portion of the 3 ’-hairpin comprises a duplex stabilizing modification. Some exemplary duplex stabilizing modifications include, but are not limited to, 2’-F nucleotides, 2’-0Me nucleotides, 2 ’-methoxy ethyl nucleotides, 2,6-diaminopurine nucleotides, 5-methyl cytidine, N4-ethyl cytidine, 5-propynyl cytidine, 5-propynyl uridine, 5-hydroxybutynl-2’- deoxyuridine, 8-aza-7-deazaguanosine, a locked nucleic acid (LNA), and / or covalent cross-linking of two strands of the duplex. In some embodiments, the stem portion comprises a covalent linkage between two nucleotides, optionally the covalent linkage is at a position other than 5 ’and 3’ of the nucleotides.

[0043] In some embodiments, a duplex stabilizing modification comprises changing an A:U base pair in the stem of the 3 ’-hairpin to a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5 or more A:U base pairs in the stem of the 3’-hairpin ton G:C base-pairs. It is known that guanine tetrads are formed by sequences rich in guanine, such as GGGGC. Thus, in order to reduce formation of guanine tetrads, G and C in the G:C base-pair replacing a A:U base-pair are positioned so that there are no more than 5, preferably no more than 4, and more preferably no more than 3 consecutive the sequence of the stem.

[0044] In some embodiments, a duplex stabilizing modification comprises a A:U base pair in the stem of the 3 ’-hairpin to a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5, 6, 7 or more A:Ubase pairs in the stem of the 3’-hairpin ton G:C base-pairs. In some embodiments, a duplex stabilizing modification comprises replacing a A:U base pair in the stem of the 5 ’-hairpin with a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5, 6, 7 or more A:U base pairs in the stem of the 3’-hairpin ton G:C base-pairs. In some embodiments, a duplex stabilizing modification comprises replacing mismatch in the stem of the 3’-hairpin with a G:C or A:U base-pair. It is known that guanine tetrads are formed by sequences rich in guanine, such as GGGGC. Thus, in order to reduce formation of guanine tetrads, G and C in the G:C base-pair replacing a A:U base-pair or a mismatch are positioned so that there are no more than 5, preferably no more than 4, and more preferably no more than 3 consecutive G in the sequence of the stem.

[0045] In some embodiments, an A:C mismatch in the stem of the 3 ’-hairpin of a wild-type or parent coRNA conserved region is replaced with a G:C base-pair. For example, the A in the mismatch is replaced by G, or the A is replaced by C and the Cis replaced by G. In some other embodiments, the A:C mismatch in the stem of the 3’-hairpin is replaced by an A:U base-pair. For example, the C in the mismatch is replaced by U, or the A is replaced by U and the Cis replaced by A.

[0046] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5 or more) A:U base-pairs present in the stem of the 3 ’-hairpin of the wild-type or parent coRNA conserved region are deleted.Middle hairpin

[0047] In various embodiments, the conserved region of coRNA comprises, at least three hairpin or stem-loop structures, where at least one of the internal hairpins (i.e., internal-hairpin) comprises a modification compared to the conserved region of the parent conserved region. For example, one or more nucleotides from the internal-hairpin of the parent or wild-type conserved region are removed or deleted. In some embodiments, 1 or at least 2 or more contiguous or non-contiguous nucleotides from the 3 ’-end hairpin of the parent or wild-type conserved region are removed or deleted in the conserved region of a coRNA described herein. For example, from 1 to about 25 contiguous or non-contiguous nucleotides from the 3 ’-end hairpin of the parent or wild-type conserved region are removed or deleted in the conserved region of a coRNA described herein. In some embodiments, a coRNA described herein comprises a conserved region where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous or non-contiguous nucleotides are removed or deleted from the ’-hairpin compared to the wild-type or parent coRNA.

[0048] It is noted that the nucleotides removed or deleted from the conserved region of the wild- type or parent coRNA’ s internal -hairpin can be replaced with a linker. For example, the deleted or the removed nucleotides can be replaced with a nucleotide or a non-nucleotide linker. In some embodiments, the nucleotides removed or deleted from the conserved region of the wild-type or parent coRNA’s internal-hairpin can be replaced with a linker are replaced with a nucleotide sequence, e.g., a single -stranded nucleic acid comprising from about comprising from about 4 nucleotides to about 15nucleotides. For example, the deleted or the removed nucleotides can be replaced with nucleotide linker comprising 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the deleted or the removed nucleotides can be replaced with a nucleotide sequence comprising 4, 5 or 6 nucleotides. In some embodiments, the deleted or the removed nucleotides are replaced with the nucleotide sequence GAAA or GAAAA. For example, the deleted or the removed nucleotides are replaced with the nucleotide sequence GAAA

[0049] The internal-hairpin comprises a double -stranded region (i.e., stem portion) and a single- stranded region (i.e., loop portion) connecting the two strands forming the stem portion of the hairpin.The stem portion of the internal-hairpin can comprise a double-stranded region comprising 4 or more nucleotide base pairs. For example, the stem portion of the internal-hairpin can comprise a double- stranded region comprising from about 3 to about 20 nucleotide base-pairs. In some embodiments, the stem portion of the internal -hairpin comprises a double-stranded region comprising from about 10 to about 30 nucleotide base-pairs. For example, the stem portion of the internal-hairpin comprises a double-stranded region comprising about 11 nucleotide base-pairs, about 12 nucleotide base-pairs, about 13 nucleotide base-pairs, about 14 nucleotide base-pairs, about 15 nucleotide base-pairs, about 16 nucleotide base-pairs, about 17 nucleotide base-pairs, about 18 nucleotide base-pairs, about 19 nucleotide base-pairs, about 20 nucleotide base-pairs, about 21 nucleotide base-pairs, about 22 nucleotide base-pairs, about 23 nucleotide base-pairs, about 24 nucleotide base-pairs, about 25 nucleotide base-pairs, about 26 nucleotide base-pairs, about 27 nucleotide base-pairs, about 28 nucleotide base-pairs, or about 29 nucleotide base-pairs.10050] In some embodiments, the stem portion of the internal-hairpin comprises a double -stranded region comprising 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide base pairs. In some preferred embodiments, the stem portion of the internal-hairpin comprises a double -stranded region comprising 18, 19, 20, 21, 22 or 23 nucleotide base pairs. For example, the stem portion of the internal-hairpin comprises a double-stranded region comprising 19 nucleotide base pairs.

[0051] In some embodiments, the stem portion of the internal-hairpin comprises two or more contiguous mismatches in the duplex region, i.e., the stem comprises one or more internal loops. For example, the stem portion of the internal-hairpin comprises two, three or four internal loops.

[0052] In some embodiments, the stem portion of the internal-hairpin comprises a double -stranded region comprising 15, 16, 17, 18, 19, 21, 22, 23, 24 or 25 nucleotide base-pairs and the stem portion further comprises 2, 3, or 4 internal loops. For example, the stem portion of the internal-hairpin comprises a double-stranded region comprising 18, 19, 20, 21, or 22 nucleotide base-pairs and the stem portion further comprises further comprises 2, 3, or 4 internal loops. In some embodiments, the stem portion of the internal -hairpin comprises a double-stranded region comprising 18, 19, 20, 21, or 22 nucleotide base-pairs and the stem portion further comprises at least one internal loop at least one (e.g., one, two, or all three) of positions 4, 14 and 17.

[0053] A pin-loop portion of the internal-hairpin can be 3 nucleotides or longer. For example, the pin-loop portion of the internal-hairpin can be from about 5 nucleotides to about 25 nucleotides. For example, the pin-loop portion of the internal-hairpin can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the pin-loop portion of the internal-hairpin is 5, 6, 7, 8, 9 or 10 nucleotides in length.

[0054] A total length of the internal-hairpin, e.g., from the first base-paired nucleotide at the 5’- end to the last base-paired nucleotide at the 3 ’-end of the hairpin, can be from about 55 nucleotides to about 85 nucleotides. For example, the internal-hairpin can comprise about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, or about 70, about 71, about 72, about 72, about 73, about 75, about 76, about 77, about 78, about 79 or about 80 nucleotides. In some embodiments, the internal-hairpin comprises 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 nucleotides. For example, the internal-hairpin comprises 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 nucleotides. In some embodiments, the internal -hairpin comprises about 66 nucleotides.

[0055] The internal-hairpin can comprise a nucleic acid modification, e.g., to stabilize the hairpin. For example, the internal-hairpin can comprise a nucleic acid modification selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof. The modification can be present at any location in the internal-hairpin. In some embodiments, the stem portion of the internal-hairpin comprises a nucleic acid modification. In some embodiments, the loop portion of the internal-hairpin comprises a nucleic acid modification. In yet some other embodiments, both the stem portion and the loop portion comprise independently selected nucleic acid modification.

[0056] In some embodiments, the stem portion and / or the loop portion of the internal-hairpin comprises a modified nucleotide selected independently from the group consisting of 2’-O-methyl (2’- OMe) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’- C-methylene (locked nucleic acid, LNA) nucleotides, 2’-O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl -carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene (Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-M0E) nucleotides, 2’-O-allyl nucleotides, 2’-C- allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-0-NMA) nucleotides, a 2'-O- dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, 2’- F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; a internal-hairpin natural or modified nucleobase; or a combination thereof. For example, the stem portion and / or the loop portion of the internal-hairpin comprises a 2’- O-methyl modified nucleotide. In another example, the stem portion and / or the loop portion of the internal -hairpin comprises a 2 ’-fluoro modified nucleotide.

[0057] The internal -hairpin can also comprise one or more (e.g., 1, 2, 3, 4, 5 or more) internal- hairpin natural or modified nucleobases. The internal-hairpin natural or modified nucleobase can be present in the stem portion or the loop portion of the internal-hairpin. In some embodiments, the stem portion of the internal -hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) internal -hairpin natural or modified nucleobases. In another embodiment, the loop portion of the internal-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) internal-hairpin natural or modified nucleobases. In yet some other embodiments, both the stem portion and the loop portion of the internal-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 ormore) independently selected internal-hairpin natural or modified nucleobases.

[0058] The internal-hairpin can also comprise at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkages. Exemplary modified intemucleoside linkages are described herein and include, but are not limited to, phosphorothioate and MMI linkages. Like other modifications, a modified intemucleotide linkage can be present in the stem portion and / or the loop portion of the internal-hairpin.

[0059] In some embodiments, the stem portion of the internal-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In another embodiment, the loop portion of the internal-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In yet some other embodiments, both the stem portion and the loop portion of the internal-hairpin comprises one or more (e.g., 1, 2, 3, 4, 5 or more) independently selected modified intemucleoside linkages (e.g., phosphorothioate and / or MMI linkages). In some embodiments, the internal-hairpin comprises a modified intemucleoside linkage (e.g., phosphorothioate and / or MMI linkage) between the stem portion and the loop portion of the internal-hairpin.

[0060] Without wishing to be bound by a theory, increasing the stability, e.g., stability of the stem of the internal -hairpin can provide increased or higher nucleic acid modification, e.g., editing activity and / or efficiency. Accordingly, in some embodiments, the stem portion of the internal-hairpin comprises a duplex stabilizing modification. Some exemplary duplex stabilizing modifications include, but are not limited to, 2’-F nucleotides, 2’-OMe nucleotides, 2 ’-methoxy ethyl nucleotides, 2,6- diaminopurine nucleotides, 5-methyl cytidine, N4-ethyl cytidine, 5-propynyl cytidine, 5-propynyl uridine, 5-hydroxybutynl-2’-deoxyuridine, 8-aza-7-deazaguanosine, a locked nucleic acid (LNA), and / or covalent cross-linking of two strands of the duplex. In some embodiments, the stem portion comprises a covalent linkage between two nucleotides, optionally the covalent linkage is at a position other than 5 ’and 3’ of the nucleotides.

[0061] In some embodiments, a duplex stabilizing modification comprises changing an A:U base pair in the stem of the internal-hairpin to a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5 or more A:U base pairs in the stem of the internal-hairpin ton G:C base-pairs. It is known that guanine tetrads are formed by sequences rich in guanine, such as GGGGC. Thus, in order to reduce formationof guanine tetrads, G and C in the G:C base-pair replacing a A:U base-pair are positioned so that there are no more than 5, preferably no more than 4, and more preferably no more than 3 consecutive the sequence of the stem.

[0062] In some embodiments, a duplex stabilizing modification comprises a A:U base pair in the stem of the internal-hairpin to a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5, 6, 7 or more A:U base pairs in the stem of the internal-hairpin ton G:C base-pairs. In some embodiments, a duplex stabilizing modification comprises replacing a A:U base pair in the stem of the 5’-hairpin with a G:C base-pair. For example, one or more, e.g., 1, 2, 3, 4, 5, 6, 7 or more A:U base pairs in the stem of the 5 ’-hairpin ton G:C base-pairs. In some embodiments, a duplex stabilizing modification comprises replacing mismatch in the stem of the 5 ’-hairpin with a G:C or A:U base-pair. It is known that guanine tetrads are formed by sequences rich in guanine, such as GGGGC. Thus, in order to reduce formation of guanine tetrads, G and C in the G:C base-pair replacing a A:U base-pair or a mismatch are positioned so that there are no more than 5, preferably no more than 4, and more preferably no more than 3 consecutive G in the sequence of the stem.

[0063] In some embodiments, a G:U base-pair in the stem of the internal -hairpin of a wild-type or parent coRNA conserved region is replaced with a G:C base-pair. For example, the U in the mismatch is replaced by C, or the U is replaced by G and the G is replaced by C.

[0064] In some embodiments of any one of the aspects described herein, one or more nucleotides in the internal-hairpin can base pair with nucleotides in another part of the sequence and form a pseudoknot structure. For example, one or more nucleotides in the pin-loop of the internal loop can base pair with nucleotides in another portion of the conserved region to from a pseudoknot structure. The nucleotides involved in forming the pseudoknot structure can be modified to stabilize the pseudoknot structure. For example, one or more A:U base-pairs or mismatches in the pseudoknot structure can be replaced with G:C base-pairs. In some embodiments, an A:U base-pair in the pseudoknot structure can be replaced with a U:U base-pair.

[0065] In some embodiments, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) nucleotides are inserted between immediately 5’- to the first nucleotide in the pin-loop of the internal hairpin that is involved in forming the pseudoknot structure.Deletions at 3 ’-end

[0066] In some embodiments, one or more nucleotides present at the 3 ’-end of the wild-type or parent coRNA conserved region, that are not involved in forming the 3 ’-hairpin are deleted. For example, 10 to about 30 nucleotides present at the 3’-end of the wild-type or parent coRNA conserved region, that are not involved in forming the 3’-hairpin are deleted. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides present at the 3 ’-end of the wild-type or parent coRNA conserved region, that are not involved in formingthe 3 ’-hairpin are deleted.Length of conserved region

[0067] In some embodiments, the coRNA comprises a conserved region that is at least 45 or more nucleotides shorter than the conserved region of the parent or wild-type coRNA. In some embodiments, the coRNA comprises a conserved region that is from about 50 to about 90 nucleotides shorter than the conserved region of the parent or wild-type coRNA. For example, the coRNA comprises a conserved region that is from about 55 to about 85 nucleotides shorter than the conserved region of the parent or wild-type coRNA. In some embodiments, the coRNA comprises a conserved region that is about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, or about 90, nucleotides shorter than the wild-type or parent coRNA. For example, the coRNA comprises a conserved region that is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides shorter than the wild-type or parent coRNA.

[0068] In some embodiments, the coRNA comprises a conserved region less than 200 nucleotides in length. For example, the coRNA comprises a conserved region comprising 195 or less, 190 or less, 185 or less, 180 or less, 175 or less, 170 or less, 165 or less, 160 or less, 155 or less, 150 or less, 145 or less, 140 or less, or 135 or less nucleotides in length. In some embodiments, the conserved region is from about 125 nucleotides to about 195 nucleotides in length. For example, the coRNA comprises a conserved region comprising from about 130 nucleotides to about 190 nucleotides in length, such as from about 135 nucleotides to about 180 nucleotides or from about 140 nucleotides to about 170 nucleotides. In some embodiments of any one of the aspects described herein, the conserved region is about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, or about 160 nucleotides in length. For example, the conserved region of coRNA is 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 nucleotides in length. In some embodiments, the conserved region of coRNA is 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, or 135 nucleotides in length.

[0069] In some embodiments of any one of the aspects described herein, the conserved region ofa coRNA described herein comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the sequence:GUCAAUAACCCGAAAGGGUUACUGAGCAGAGCAGUGACAUGUCAU UCGCCGCGGGGUGAUUCCAAGCUCCGCGCUCCGGCUAGACAUGCC CAUGCUAUGGAAACUUUAACGGUGAAAACCGGCUUACAACAAA (SEQ ID NO: 10)

[0070] A conserved region consisting of SEQ ID NO: 10 is also referred to a M71 conserved region. In some embodiments, the conserved region of a coRNA comprises the sequence of M71 conserved region, wherein the conserved region further comprises at least one, at least two, at least three, at least four, at least five, at least at least six, seven, at least eight, at least nine or at modifications from Table 19 in the Examples, where each T is replaced by U.Guide sequence (spacer, variable region)

[0071] In some embodiments, guide nucleic acid, e.g., coRNA comprises a guide sequence, also referred to as a spacer or spacer sequence herein. In some embodiments, the guide sequence comprises a nucleotide sequence that is complementary to a portion of a target nucleic acid. The guide sequence can be referred to as a “cr sequence” herein. Generally, the spacer or guide sequence comprises a sequence of at least 10 nucleotides that is complementary to a target sequence. In some embodiments, the spacer or guide sequence comprises a sequence of from 10 nucleotides to 50 nucleotide that is complementary to a target sequence. For example, the spacer or guide sequence comprises a sequence of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence.

[0072] The spacer and the conserved region of the guide nucleic acid, e.g., coRNA can be located in any preferred position to each other. In some embodiments, the spacer or guide sequence is at 5’- end of the conserved region of the guide nucleic acid, e.g., coRNA. In other words, 3 ’-end of the spacer is lined to the 5’-end of the conserved region of the guide nucleic acid, e.g., coRNA. The spacer and the conserved region can be linked directly (e.g., by a bond or modified or unmodified intemucleoside linkage) or by a nucleotide sequence comprising from 1 to 25 nucleotides or by a non-nucleotide linker. In some preferred embodiments, the 3 ’-end of the spacer is linked directly to the 5 ’-end of the conserved region by a modified (e.g., phosphorothioate, imidp or MMI linkage) or unmodified (e.g., phosphodiester) intemucleoside linkage)

[0073] The nucleotide sequence of the spacer (i.e., guide sequence) can be chosen to direct site- specific binding to a target sequence of a target nucleic acid. The spacer can comprise an engineered heterologous sequence. Generally, the spacer is from about 10 to about 50 nucleotides in length. In some embodiments, the spacer is at least 15 nucleotides in length. For example, the spacer is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length. In some embodiments, the spacer is from about 15 to about 17 nucleotides, from about 18 to about 20 nucleotides, from about 21 to about 23 nucleotides, from about 24 to about 26 nucleotides, from about 27 to about 29 nucleotides, from about 30 to about 32 nucleotides, from about 33 to about 35 nucleotides, from about 36 to about 38 nucleotides, from about 39 to about 41 nucleotides, from about 42 to about 44 nucleotides, from about 45 to about 47 nucleotides, or from about 48 to about 50 nucleotides in length. In some embodiments, the spacer is from 15 to 17 nucleotides, from 15 to 23 nucleotides, from 15 to 30 nucleotides, from 16 to 22 nucleotides, from 17 to 20 nucleotides, from 20 to 24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides), from 23 to 25 nucleotides (e.g., 23, 24, or 25 nucleotides), from 24 to 27 nucleotides, from 27 to 30 nucleotides, from 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 40, or 45 nucleotides), from 30 or 35 to 40 nucleotides, from 41 to 45 nucleotides, from 45 to 50 nucleotides in length. In some embodiments, the spacer is 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 40, 41, 42, 43, 44, 45, 46, 47 48, 49, or 50 nucleotides in length. In some preferred embodiments, the spacer is from 16 to 23 nucleotides (e.g., 16, 17, 18, 19, 21, 22 or 23 nucleotides) in length. For example, the spacer is 19, 20 or 21 nucleotides in length.

[0074] In some embodiments, the spacer or guide sequence comprises from about 10 to about 100 nucleotides and a sequence of at least 10 nucleotides that is complementary to a target sequence. For example, the spacer or guide sequence comprises from about 10 to about 100 nucleotides and a sequence of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence.

[0075] In some embodiments, the spacer or guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long and comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence.

[0076] To reduce off-target interactions, e.g., to reduce the spacer interacting with a non-target sequence, mutations can be introduced so that the engineered, non-natural DNA and RNA targeting complexes described herein can distinguish between target and off-target sequences that have greater than 80%, 85%, 90%, or 95% complementarity. In some embodiments, the degree of complementarity is from 80% to 95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (for example, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2, or 3 mismatches). Accordingly, in some embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence is greater than 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%. In some embodiments, the degree of complementarity between the spacer sequence and the target nucleic acid is 100%. Degree of complementarity between differentnucleic acid sequence can be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman- Wunsch algorithm, algorithms based on the Burrows- Wheel er Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0077] For all embodiments of the systems and methods described herein, it is known in the field that complete complementarity is not required for hybridization or binding as described herein, provided that there is sufficient complementarity to be functional. Accordingly, in some embodiments, the nucleotide sequence of the spacer comprises at least one (e.g., 2, 3, 4 or 5) mismatch with the target nucleic acid. In some embodiments, the spacer sequence is designed to have a stretch of two or more adjacent mismatching nucleotides with the target nucleic acid.

[0078] Without wishing to be bound by a theory, modulations of cleavage efficiency can be exploited by introducing mismatches, e.g., one or more mismatches, such as 1 or 2 mismatches between a spacer sequence and a target sequence, including the position of the mismatch along the spacer / target. The more central (i.e., not at the 3’ or 5’ ends) a mismatch, e.g., a double mismatch, is located; the more cleavage efficiency is affected. Accordingly, by choosing mismatch positions along the spacer sequence, cleavage efficiency can be modulated. For example, if less than 100% cleavage of targets is desired (e.g., in a cell population), 1 or 2 mismatches between spacer sequence and target sequence can be introduced in the spacer sequences.

[0079] In some embodiments, the guide nucleic acid comprises a direct repeat (DR) sequence linked to the guide sequence. The direct repeat sequence comprises at least one hairpin or stem loop structure . Generally, the direct repeat sequence has a minimum length of 16 nucleotide s and one hairpin or stem loop. In some embodiments, the direct repeat sequence has a length longer than 16 nucleotides, preferably more than 17 nucleotides, and has two or more hairpin or stem loops. In some embodiments, the hairpin or the stem loop structure comprises at least 5, preferably 7-20 nucleotides. The direct repeat sequence can be 3’ of the spacer (guide sequence). Alternatively, the direct repeat sequence can be 5’ of the spacer (guide sequence). In some embodiments, the spacer is flanked by a direct repeat sequence at its 5’ and 3’ ends. It is noted that a guide nucleic acid comprising a spacer flanked by a direct repeat sequence at its 5’ and 3’ ends (DR-spacer-DR) structure is typical of precursor crRNA (pre-crRNA). In some embodiments, the guide nucleic acid comprises a truncated direct repeat sequence and a spacer sequence, which is typical of processed or mature crRNA.

[0080] The direct repeat can be at least 10 nucleotides in length. For example, the direct repeat can be at least 11 nucleotides, or at least 12 nucleotides, or at least 13 nucleotides, or at least 14 nucleotides, or at least 15 nucleotides, or at least 16 nucleotides, or at least 17 nucleotides, or at least18 nucleotides, or at least 19 nucleotides, or at least 30 nucleotides in length. In some embodiments, the direct repeat comprises the sequence NACACC, proximal to the spacer, where N is G or T.

[0081] The guide nucleic acid can further include a tracr sequence for complexing with the protein effector polypeptide. The term “tracr sequence”, as used herein, can generally refer to a nucleic acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and / or sequence similarity to a wild type exemplary tracrRNA sequence. In some embodiments of any one of the aspects described herein, the tracr sequence comprises one or more, e.g., 1, 2, 3, 4 or more hairpins or stem loops. In some embodiments, the tracr sequence comprises a sequence predicted to comprise at least two hairpins comprising less than 5 base-paired ribonucleotides. The spacer and the tracr sequences can be linked to each other via a hairpin or stem loop structure. The tracr sequence can be 5 or more nucleotides in length. For example, the tracr sequence can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.

[0082] The guide nucleic acid can be single-stranded or double-stranded. In some embodiments, the guide nucleic acid is single polynucleotide chain, and optionally comprises double-stranded regions. Guide nucleic acid that comprise a single polynucleotide chain can be referred to as a “single guide nucleic acid.” It is noted that when the guide nucleic acid is a single polynucleotide chain, the spacer and the tracr sequence can be located in any preferred position to each other. For example, the spacer can be located 5 ’ of the tracr sequence . Alternatively, the spacer can be located 3 ’ of the tracr sequence . In some preferred embodiments, the spacer is 5’ of the tracr sequence.

[0083] In some embodiments, the spacer and the tracr sequence of the guide nucleic acid are present in separate polynucleotide chains. Optionally, the separate polynucleotides chains are partially hybridized to each other. Guide nucleic acid that comprise two polynucleotide chains can be referred to as a “double guide nucleic acid.” Generally, the 3 ’-end of the polynucleotide chain comprising the spacer hybridized with the 5 ’-end of the polynucleotide chain comprising the tracr sequence. The polynucleotide chain comprising the spacer is also referred to as “crRNA” herein, and the polynucleotide chain comprising the tracr sequence is also referred to as “tracrRNA” herein.

[0084] In some embodiments, the spacer or guide sequence is linked to a direct repeat and the tracr sequence is linked to an anti -repeat sequence. The direct repeat and anti-repeat sequences are substantially complementary to each other and can hybridize with each other to form a double -stranded region. The double-stranded region can be at least 8, or at least 9, or at least 10, or at least 11, or at least 12 base-pairs in length.

[0085] The guide nucleic acid can also include one or more protein binding domains, e.g., for binding with or recruiting one or more gene effectors, gene activators, or gene repressors. Generally, the protein binding domain of the guide nucleic acid comprises a scaffold that is capable of binding with a protein. In some embodiments, the protein binding domain can be an aptamer. Aptamers are oligonucleotide or peptide molecules that can bind to a specific target molecule. The aptamers can bespecific to gene effectors, gene activators, or gene repressors. In some embodiments, the aptamers can be specific to a protein, which in turn is specific to and recruits / binds to specific gene effectors, gene activators, or gene repressors. The effectors, activators, or repressors can be present in the form of fusion proteins. In some embodiments, the guide nucleic acid comprises two or more aptamer sequences that are specific to the same adaptor proteins. In some embodiments, the two or more aptamer sequences are specific to different adaptor proteins. The adaptor proteins can include, but are not limited to, MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ɸCb5, ɸCb8r, ɸCb12r, ɸCb23r, 7s, and PRR1. Accordingly, in some embodiments, the aptamer is selected from binding proteins specifically binding any one of the adaptor proteins as described herein. In some embodiments, the aptamer sequence is a MS2 binding loop, Qβ binding loop, or PP7 binding loop. A detailed description of aptamers can be found, e.g., in Nowak et al., “Guide RNA engineering for versatile Cas9 functionality,” Nucl. Acid. Res., 2016 Nov l6;44(20):9555-9564; and WO2016205764, which are incorporated herein by reference in their entirety.

[0086] In an embodiment, the guide nucleic acid further comprises an extension to add an RNA template. In some embodiments, a “protector nucleic acid” can be hybridized to a sequence of the guide nucleic acid, wherein the “protector nucleic acid” is nucleic acid strand complementary to the 3’ end of the guide nucleic acid to thereby generate a partially double-stranded guide nucleic acid. In some embodiments, protecting mismatched bases (i.e., the bases of the guide nucleic acid which do not form part of the guide sequence) with a perfectly complementary protector sequence decreases the likelihood of a target sequence binding to the mismatched basepairs at the 3’ end of the guide nucleic acid. In some embodiments, additional sequences comprising an extended length can also be present within the guide nucleic acid such that the guide nucleic acid comprises a protector sequence within the guide nucleic acid molecule. This “protector sequence” ensures that the guide nucleic acid molecule comprises a “protected sequence” in addition to an “exposed sequence” (comprising the part of the guide nucleic acid sequence hybridizing to the target sequence). In some embodiments, the guide nucleic acid is modified by the presence of the protector nucleic acid to comprise a secondary structure such as a hairpin. Advantageously there are three or four to thirty or more, e.g., about 10 or more, contiguous base pairs having complementarity to the protected sequence, the guide nucleic acid sequence or both. It is advantageous that the protected portion does not impede thermodynamics of the protein effector polypeptide and related system interacting with its target. By providing such an extension including a partially double stranded guide nucleic acid, the guide nucleic acid is considered protected and results in improved specific binding of the protein effector polypeptide / guide nucleic acid, while maintaining specific activity.

[0087] In some embodiments, the guide nucleic acid structure comprises a sequence predicted to comprise a hairpin consisting of a stem and a loop, wherein the stem comprises at least 10, at least 12 or at least 14 base-paired ribonucleotides, and an asymmetric bulge within 4 base pairs of the loop.

[0088] In some embodiments, the guide nucleic acid comprises a sequence predicted to comprise a hairpin with an uninterrupted base-paired region comprising at least 8 nucleotides of a guide sequence and at least 8 nucleotides of a tracr sequence, and wherein the tracr sequence comprises, from 5’ to 3’, a first hairpin and a second hairpin, wherein the first hairpin has a longer stem than the second hairpin.

[0089] The guide nucleic acids can be chemically synthesized or can be generated as components of inducible systems. The inducible nature of the systems allows for spatiotemporal control of gene editing or gene expression. In some embodiments, the stimuli for the inducible systems include, e.g., electromagnetic radiation, sound energy, chemical energy, and / or thermal energy. For example, the transcription of guide nucleic acids can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone inducible gene expression systems (e.g., ecdysone inducible gene expression systems), and arabinose- inducible gene expression systems. Other examples of inducible systems include, e.g., small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.), light inducible systems (Phytochrome, LOV domains, or cryptochrome), or Light Inducible Transcriptional Effector (LITE). These inducible systems are described, e.g., in WO 2016205764 and US 8795965, both of which are incorporated herein by reference in their entirety.

[0090] The architecture of multiple RNA guides is known in the art. See, for example, International Publication Nos. WO 2014 / 093622 and WO 2015 / 070083, the entire contents of each of which are incorporated herein by reference). Thus, in some embodiments, the engineered, non-natural DNA and RNA targeting complexes described herein include multiple guide nucleic acids (e.g., two, three, four, five, six, seven, eight, or more guide nucleic acids). Sequences for RNA guides from multiple CRISPR systems are known in the art and can be searched using public databases (see, e.g., Grissa et al. (2007) Nucleic Acids Res. 35 (web server issue): W52-7; Grissa et al. (2007) BMC Bioinformatics 8: 172; Grissa et al. (2008) Nucleic Acids Res. 36 (web server issue): W145-8; and Moller and Liang (2017) PeerJ 5: e3788; see also the CRISPR database available at: crispr.i2bc.paris- saclay.fr / crispr / BLAST / CRISPRsBlast.php; and MetaCRAST available atgithub.com / molleraj / MetaCRAST).

[0091] In some embodiments, an engineered, non-natural DNA and RNA targeting complexes described herein comprises two different guide nucleic acids targeting a first region and a second region in a target nucleic acid. In some embodiments, the first region is 5’ to the second region. In some other embodiments, the first region is 3’ to the second region.

[0092] In some embodiments, two different guide nucleic acids can be encoded by a single polynucleotide. For example, the polynucleotide sequence encoding a guide nucleic acid comprises a CRarray comprising two or more guide sequences.

[0093] The guide nucleic acid sequences can be modified in a manner that allows for formation of a complex comprising the protein effector polypeptide and the guide nucleic acid, and successfulbinding to the target, while at the same time not allowing for successful nuclease activity. These modified guide sequences are referred to as “dead guides” or “dead guide sequences.” These dead guides or dead guide sequences can be catalytically inactive or conformationally inactive with regards to nuclease activity. Dead guide sequences are typically shorter than respective guide sequences that result in active target (e.g., RNA or DNA) cleavage. In some embodiments, dead guides are 5%, 10%, 20%, 30%, 40%, or 50%, shorter than respective guide nucleic acids that have nuclease activity. Dead guide sequences of guide nucleic acids can be from 13 to 15 nucleotides in length (e.g., 13, 14, or 15 nucleotides in length), from 15 to 19 nucleotides in length, or from 17 to 18 nucleotides in length (e.g., 17 nucleotides in length). Thus, in one aspect, the disclosure provides engineered, non-natural DAN and RNA targeting system or composition comprising a functional protein effector polypeptide as described herein, and a guide nucleic acid, wherein the guide nucleic acid includes a dead guide sequence whereby the guide nucleic acid is capable of hybridizing to a target sequence such that the complex is directed to a genomic locus of interest, e.g., in a cell without detectable cleavage activity. A detailed description of dead guides is described, e.g., in WO 2016094872, which is incorporated herein by reference in its entirety.

[0094] In some embodiments of any one of the aspects described herein the guide RNA is an coRNA described herein.

[0358] Also provided herein are DNA and RNA targeting systems comprising an IscB protein (or a fusion protein) described herein and a guide RNA nucleic acids (e.g., a guide RNA or coRNA). Also provided herein are DNA and RNA targeting systems comprising: guide RNA nucleic acids (e.g., a guide RNA or coRNA) described herein and a protein effector polypeptide.

[0359] In some embodiments, a DNA and RNA targeting complex described herein exhibits a lower or decreased off-target activity, i.e., modification of a non-target sequence, as compared to the off-target activity of a complex comprising a wild-type IscB and wild-type gRNA. For example, the off-target activity of a DNA and RNA targeting complex described herein, is at least at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold lower than the off-target activity of a complex comprising a wild-type IscB and wild-type gRNA.Conservative variants

[0360] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0361] The term “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined or native amino acid sequence with a different “replacement” amino acid. A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for...

Claims

CLAIMSWhat is claimed is:

1. An IscB protein (protein effector polypeptide) comprising:(a) an amino acid sequence that is at least 80% identical to the amino acid sequence of anIscB as provided by any one of SEQ ID NO: 2, wherein the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations in an amino acid residue selected from the group consisting of amino acid residues 18, 34, 36, 37, 60, 66, 85, 97, 99, 100, 101, 104, 109, 112, 118, 136, 137, 155, 156, 157, 166, 168, 190, 204, 204, 205, 206, 208, 212, 214, 216, 218, 219, 229, 229, 230, 233, 233, 234, 234,238, 239, 241, 242, 245, 246, 248, 252, 254, 255, 256, 257, 258, 279, 280, 291, 293,298, 303, 304, 306, 307, 312, 319, 323, 328, 329, 331, 332, 338, 341, 353, 360, 368,372, 376, 377, 378, 379, 380, 381, 382, 384, 385, 386, 388, 390, 391, 393, 405, 406,408, 409, 410, 412, 413, 416, 417, 418, 419, 421, 421, 423, 425, 437, 439, 455, 462,463, 478, 479, 533, 536, 564 and 565 of the amino acid sequence of SEQ ID NO: 2 (pEDIT071), and provided that, if the IscB comprises a single mutation, it is not Q60I, N66H, I85R, I85K, E137R, P405R, D413K, E409R or combination thereof; or(b) an amino acid sequence that is at least 80% identical to the amino acid sequence of an IscB as provided by any one of SEQ ID NO: 8, wherein the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations in an amino acid residue selected from the group consisting of amino acid residues30, 46, 48, 49, 72, 78, 97, 109, 111, 112, 113, 116, 121, 124, 130, 148, 149, 167, 168, 169, 178, 180, 202, 216, 216, 217, 218, 220, 224, 226, 228, 230, 231, 241, 241, 242,245, 245, 246, 246, 250, 251, 253, 254, 257, 258, 260, 264, 266, 267, 268, 269, 270,291, 292, 303, 305, 310, 315, 316, 318, 319, 324, 331, 335, 340, 341, 343, 344, 350,353, 365, 372, 380, 384, 388, 389, 390, 391, 392, 393, 394, 396, 397, 398, 400, 402,403, 405, 417, 418, 420, 421, 422, 424, 425, 428, 429, 430, 431, 433, 433, 435, 437,449, 451, 467, 474, 475, 490, 491, 545, 548, 576, and 577 of the amino acid sequence of SEQ ID NO: 8 (pEDIT071 +NLS), and provided that, if the IscB comprises a single mutation, and optionally, if the IscB comprises a single mutation, the mutation is not Q72I, H78H, I97R, I97K, E149R, P417R, E421R, D425K, or combination thereof2. The IscB protein of claim 1, wherein the amino acid sequence of the IscB protein comprises: (a) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting ofvariant C18R, A34R, D36R, T37R, Q60I, N66H, I85X, I85X, M99X, A101X, A104R, E109X, A112K, R118K, S136X, E137R, I155X, L156X, T157X, T166X, L168K, L190K, G204X, N205X, I206K, K208R, K212R, K214R, N216R, K218R, D219X, A229X, K230R, L233X, P234X, P238X, F239X, K241R, E242X, A245X, E246X, E248R, T252X, E254X, K255R, S256X, T257X, F258X, S279R, G280R, L291X, K293R, Q298K, E3O3X, M304K, K306R, R307K, C312X, T319K, E323X, E328R, Q329R, D331R, G332R, G338R, S341R, R353K, A360X, K368R, L372R, D376R, A377R, D378R, T379R, A380R, E381R, S382R, T384R, E385R, M386R, T388R, L390X, M391X, K393R, P405K, K406R, V408R, E409X, T410R, A412R, D413R, P416R, G417R, H418K, F419R, V421X, N423R, W425K, E437R, D439X, E455R, E462R, P463R, I533K, T536R, H478X, H479X, I564X, and T565X of the amino acid sequence provided in SEQ ID NO: 2, wherein X is any amino acid, and optionally, if the IscB comprises a single mutation, it is not Q60I, N66H, I85R, I85K, E137R, P405R, D413K, E409R or combination thereof; or(b) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected independently from the group consisting of variant C30R, A46R, D48R, T49R, Q72I, N78H, I97X, I109X, Mi l IX, A1113X, A116R, E121X, A124K, R130K, S148X, E149R, I167X, L168X, T169X, T178X, L180K, L202K, G216X, N217X, I218K, K2220R, K224R, K2226R, N228R, K230R, D231X, A241X, K242R, L245X, P246X, P250X, F251X, K253R, E254X, A257X, E258X, E260R, T264X, E266X, K267R, S268X, T269X, F270X, S291R, G292R, L303X, K2305R, Q310K, E315X, M316K, K318R, R319K, C324X, T331K, E335X, E340R, Q343R, G344R, G350R, S353R, R365K, A372X, K380R, L384R, D388R, A389R, D390R, T391R, A392R, E393R, S394R, T396R, E397R, M398R, T400R, L402X, M403X, K3405R, P417K, K418R, V420R, E421X, T422R, A424R, D425R, P428R, G429R, H430K, F431R, V433X, N435R, W437K, E449R, D451X, E467R, E474R, P475R, I545K, T548R, H490X, H491X, I576X, and T577X of the amino acid sequence SEQ ID NO: 8, wherein X is any amino acid, and optionally, if the IscB comprises a single mutation, the mutation is not Q72I, H78H, I97R, I97K, E149R, P417R, E421R, D425K, or combination thereof3. The IscB protein of claim 1, wherein the amino acid sequence of the IscB protein comprises:(a) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of variant C18R, A34R, D36R, T37R, Q60I, N66H, I85X, I85X, M99X, AIOIX, A104R, E109X, A112K, R118K, S136X, E137R, I155X, L156X, T157X, T166X, L168K, L190K, G204X, N205X, I206K, K208R, K212R, K214R, N216R, K218R, D219X, A229X, K230R, L233X, P234X, P238X, F239X, K241R, E242X, A245X, E246X,E248R, T252X, E254X, K255R, S256X, T257X, F258X, S279R, G280R, L291X, K293R, Q298K, E303X, M304K, K306R, R307K, C312X, T319K, E323X, E328R, Q329R, D331R, G332R, G338R, S341R, R353K, A360X, K368R, L372R, D376R, A377R, D378R, T379R, A380R, E381R, S382R, T384R, E385R, M386R, T388R, L390X, M391X, K393R, P405K, K406R, V408R, E409X, T410R, A412R, D413R, P416R, G417R, H418K, F419R, V421X, N423R, W425K, E437R, D439X, E455R, E462R, P463R, I533K, T536R, H478N, H479N, I564K, I564H, I564N, I564Q, I564C, I564M, I564T, I564D, T565R, T565K, T565H, T565C, T565D, T565G, H478C, H478P, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2, and optionally, if the IscB comprises a single mutation, it is not Q60I, N66H, I85R, I85K, E137R, P405R, D413K, E409R or combination thereof; or(b) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected independently from the group consisting of C30R, A46R, D46R, T49R, Q72I, N78H, I97R, I97K, D109R, D109K, Ml HR, Mi l IK, A112K, A113R, A113K, A116R, E121R, E121K, A124K, R130K, S148R, S148K, E149R, E149K, I167R, I167K, L168R, L168K, T169R, T169K, T178R, T178K, L180K, L1202K, G216R, G216K, N217R, N217K, I218K, K220R, K224R, K2226R, N228R, K230R, D231R, D231K, A241R, A241K, K242R, L245R, L245K, P246R, P246K, P250R, P250K, F251R, F251K, K253R, E254R, E2454K, A257R, A257K, E258R, E2458K, E260R, T264R, T264K, E266R, E266K, K267R, S268R, S268K, T2569R, T2569K, F270R, F270K, S291R, G292R, L303R, L303K, K305R, Q310K, E315R, E315K, M316K, K318R, R319K, C324R, C324K, T331K, E335R, E335K, E340R, Q341R, D343R, G344R, G350R, S353R, R365K, A372R, A372K, K380R, L384R, D388R, A389R, D390R, T391R, A392R, E393R, S394R, T396R, E397R, M398R, T400R, L402R, L402K, M4031R, M403K, K405R, P417K, K419R, V420R, E421K, E421R, T422R, A424R, D425R, D425K, P428R, G429R, H430K, F431R, V433R, V433K, N435R, W437K, E449R, D451R, D451K, E4567R, E474R, P475R, I545K, T548R, H490N, H491N, I576K, I576H, I576N, I576Q, I576C, I576M, I576T, I5676D, T577R, T577K, T577H, T577C, T577D, T577G, H490C, H490P, H491C, and H491M of the amino acid sequence provided in SEQ ID NO:

8. Optionally, if the IscB comprises a single mutation, the mutation is not Q72I, H78H, I97R, I97K, E149R, P417R, E421R, D425K, or combination thereof4. The IscB protein of any one of claims 1-3, wherein the IscB protein comprises: (i) at least E409R, D413K, 533K, and at least one other mutation listed in Table A; or (ii) at least E421R, D425K, 545K, and at least one other mutation listed in Table A2.

5. The IscB protein of any one of claims 1-3, wherein the IscB protein comprises: (i) at least E409R, D413K, 533K E137K, N66H, A100K, T536R, and at least one other mutation listed inTable A; or (ii) at least E421R, D425K, 545K E149K, N78H, Al 12K, T548R, and at least one other mutation listed in Table A2.

6. The IscB protein of any one of claims claim 1-5 comprising: an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of an IscB as provided by SEQ ID NO: 2 or 8.

7. The IscB protein of any one of claims 1-6, wherein the IscB exhibits an increased activity on a target sequence that does not comprise the canonical TAM (5’-ATAAA-3’) at its 3' end as compared to IscB as provided by SEQ ID NO: 2 or 8.

8. The IscB protein of any one of claims 1-7, wherein the IscB protein exhibits an activity on a target sequence having a 3 ' end that is not directly adj acent to the canonical TAM sequence (5 ’ - ATAAA-3’) that is at least 5 -fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of IscB as provided by SEQ ID NO: 2 or 8 on the same target sequence.

9. The IscB protein of any one of claims 6-8, wherein the activity is measured by a nuclease assay, a deamination assay, or a transcriptional activation assay.

10. The IscB protein of any one of claims 1-3, wherein the IscB protein comprises: (i) a E409R, D413K, and 533K mutations of the amino acid sequence provided in SEQ ID NO: 2; or (ii) a E421R, D425K, and 545K mutations of the amino acid sequence provided in SEQ ID NO: 8.

11. The IscB protein of any one of claims 1-3 or 10, wherein the IscB protein comprises: (i) a E137K, N66H, A100K, and / or T536R mutation of the amino acid sequence provided in SEQ ID NO: 2; or (ii) a E149K, N78H, Al 12K, and / or T548R mutation of the amino acid sequence provided in SEQ ID NO: 8.

12. The IscB protein of claim 11, wherein the IscB protein comprises: (i) E409R, D413K, 533K E137K, N66H, A100K, and T536R mutations of the amino acid sequence provided in SEQ ID NO: 2; or (ii) E421R, D425K, 545K E149K, N78H, Al 12K, and T548R mutations ofthe amino acid sequence provided in SEQ ID NO: 8.

13. The IscB protein of any one of claims 1-11, wherein the IscB exhibits an increased activity on a target sequence comprising a TAM sequence selected from the group consisting of RTAAA, ATRAA, ATRMM, ATRA, ATRRA, ATARA, ATRRAC, ATAAAC, ATARM, ATRAM, ATRRM, ATRMA, ATANA, ATRR, ATR, ATGAC, ATAA, ATAR, ATGMC, ATGAA, and ATRAR at its 3' end as compared to IscB as provided by SEQ ID NO: 2 or 8, wherein R is a purine and M is A or C.

14. The IscB protein of claim 13, wherein the IscB comprises: (i) at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of variant H478X, H479N, I564X, T565X, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2, whereinX is any amino acid; or (ii) at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of variant H490X, H491N, I576X, T577X, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 8, wherein X is any amino acid15. The IscB protein of claim 14, wherein the IscB comprises at least one, at least two, at least 3 or at least 4 mutations selected from the group consisting of variant H478N, H479N, I564K, I564H, I564N, I564Q, I564C, I564M, I564T, I564D, T565R, T565K, T565H, T565C, T565D, T565G, H478C, H478P, H479C, and H479M of the amino acid sequence provided in SEQ ID NO: 2; or (ii) H490N, H491N, I576K, I576H, I576N, I576Q, I576C, I576M, I576T, I576D, T577R, T577K, T577H, T577C, T577D, T577G, H490C, H490P, H491C, and H491M of the amino acid sequence provided in SEQ ID NO: 8.

16. The IscB protein of any one of claims 1-15, wherein the IscB exhibits an increased activity on a target sequence comprising a TAM sequence is ATR.

17. The IscB protein of claim 13, comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 amino sequences swap at position 560-571 of SEQ ID NO: 2, or at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 amino sequences swap at position 572-583 of SEQ ID NO: 8.

18. The IscB protein of claims 17, comprising at least 3, at least 4, at least 5, or at least 6 amino sequences swap at position Q563-K568 of SEQ ID NO: 2 or at least 3, at least 4, at least 5, or at least 6 amino sequences swap at position Q575-K580 of SEQ ID NO: 8.

19. The IscB protein of claim 18, comprising six amino sequences swap at position Q563-K568 of SEQ ID NO: 2 or at position is Q575-K580 of SEQ ID NO: 8 is selected from QTSGGR, RSSGGK, QQSKGR, QQGRGR, QQNKGR, and QKDKGA.

20. The IscB protein of claim 19, wherein the IscB exhibits an increased activity on a target sequence comprising a TAM sequence is ATR.

21. The IscB protein of claim 17, comprising at least 7, at least 8, at least 9, at least 10 or at least 11 amino sequences swap at position 560-571 of SEQ ID NO: 2, or at position 572-583 of SEQ ID NO: 8.

22. The IscB protein of claim 18, comprising the amino acid sequence MSGQHCHGAYLR,LSGNSHYGQYYM, MTGQHCHGNYFR, MTGQQNKGDRYF, KSGQLTNGQYFR, LKGNACNGAYFH, SSGTHAKSYFR, LGGQKNNGVYFY, VTGSSGAGKYVR,VRSVHSYGKYVR, VQGIKNRGRYLH, LRGTSSYGKTVL, VKGVFNYGKWVR,VKGKHNDRWI, VKGVQNRGKYIK, IKGSHCNGSRLI, SGGVMSYGRYIT,SKGNKNKGKSIL, AIGTHCKGKRVI, VKGVFNRGTWVR, CKGMHSYGRYVL,IGGTQNKGRYAI, or TKGTHCKGTRIM at position M560-R571 of SEQ ID NO: 2.

23. The IscB protein of claim 20, wherein the IscB exhibits an increased activity on a target sequence comprising a TAM sequence is ATRR.

24. The IscB protein of any one of claims 1-13, wherein the IscB exhibits an increased activity on a target sequence comprising a TAM sequence is RTAAA.

25. The IscB protein of claim 24, wherein the IscB comprises a mutation at position 478 and / or 479 of the amino acid sequence provided in SEQ ID NO: 2, or at position 490 and / or 491 of the amino acid sequence provided in SEQ ID NO:

226. The IscB protein of claim 25, wherein the IscB comprises: (i) a mutation H478N, H479N, H478C and / or H478P of the amino acid sequence provided in SEQ ID NO: 2; or (ii) a mutation H490N, H491N, H490C and / or H490P of the amino acid sequence provided in SEQ ID NO: 8.

27. The IscB protein of any one of claims 1-26, wherein the IscB protein exhibits lower off-target activity as compared to an off-target activity of the IscB domain as provided by SEQ ID NO: 2 or 8.

28. The IscB protein of any one of claims 1 -27, wherein the IscB protein further comprises a nuclear localization signal (NLS) sequence.

29. The IscB protein of claim 28, wherein the NLS comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any one of the NLS sequences show in in Table C, or a homolog or ortholog thereof.

30. The IscB protein of claim 28 or 29, wherein the NLS is linked to N-terminal of the IscB protein.

31. The IscB protein of claim 28 or 29, wherein the NLS is linked to C-terminal of the IscB protein.

32. A fusion protein comprising (i) the IscB protein of any one of claims 1-31 and (ii) an effector domain.

33. The fusion protein of claim 32, wherein the effector domain is a domain that comprises nuclease activity, nickase activity, recombinase activity, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activation activity, or transcriptional repression activity.

34. The fusion protein of claim 32 or 33, wherein the effector domain is a nucleic acid editing domain.

35. The fusion protein of claim 32 or 33, wherein effector domain comprises a deaminase domain, a reverse transcriptase, or a functional fragment thereof.

36. The fusion protein of claim 35 wherein the deaminase domain is a cytidine deaminase domain.

37. The fusion protein of claim 36, wherein the cytidine deaminase domain is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.

38. The fusion protein of claim 36 or 37, wherein the cytidine deaminase domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the cytidine deaminase domain of any one of the sequences in Table C.

39. The fusion protein of claim 35, wherein the deaminase domain is an adenosine deaminase domain.

40. The fusion protein of claim 35, further comprising a second adenosine deaminase.

41. The fusion protein of claim 36, wherein the first adenosine deaminase domain and the second adenosine deaminase domain comprises an ecTadA domain, or variant thereof.

42. The fusion protein of claim 36 or 37, wherein the first adenosine deaminase domain and the second adenosine deaminase domain comprise independently an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the cytidine deaminase domain of any one of the sequences in Table DI.

43. The fusion protein of any one of claims 32-42, wherein the fusion protein further comprises a uracil glycosylase inhibitor (UGI) domain.

44. The fusion protein of claim 43, wherein the UGI domain comprises an amino acid sequence having is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any one of the sequences in Table E.

45. The fusion protein of any one of claims 43-44, wherein the fusion protein further comprises a second UGI domain.

46. A coRNA comprising a conserved portion of a coRNA (parent coRNA) comprising one or more stem-loop structures, wherein at least one of the stem-loop structures comprises a modification, and wherein a conserved portion of the coRNA is shorter than the conserved portion of the parent coRNA and is capable of binding with a transposon family encoded nuclease or a homolog or ortholog thereof.

47. The coRNA of claim 46, wherein the conserved region of the coRNA comprises at least two stem-loop structures and wherein said modification is in a stem-loop structure near to the 5’- end (5 ’-stem -loop) of the conserved region coRNA.

48. The coRNA of claim 47, wherein said modification comprises deletion of from about 3 to about 46 nucleotides from the 5 ’-stem -loop.

49. The coRNA of claim 47 or 48, wherein said modification comprises deletion of 3, 7, 11, 15, 18,22, 26, 30, 34, 37 or 41 nucleotides from the 5 ’-stem-loop.

50. The coRNA of claim 4, wherein said modification comprises deletion of 15, 18, 30, 37 or 41 nucleotides from the 5 ’-stem-loop.

51. The coRNA of claim 47 or 48, wherein said modification comprises deletion of 8, 12, 16, 20,23, 27, 31, 35, 39, 42 or 46 nucleotides from the 5 ’-stem -loop.

52. The coRNA of claim 51, wherein said modification comprises deletion of 20, 23, 35, 42 or 46 nucleotides from the 5 ’-stem-loop.

53. The coRNA of any one of claims 47-52, wherein nucleotides at position 12-57, 14-55, 16-54, 18-52, 20-50, 22-48, 24-46, 26-45, 28-43, 30-41 or 32-39 are deleted (counting from 5 ’-end, e.g., 5 ’-end of the conserved region of the parent coRNA).

54. The coRNA of claim 53, wherein nucleotides at position 12-57, 14-55, 18-52, 24-46, or 26-45 are deleted (counting from 5 ’-end, e.g., 5 ’-end of the conserved region of the parent coRNA).

55. The coRNA of any one of claims 47-54, wherein the deleted nucleotides are replaced with a linker (e.g., a loop portion or pin-loop).

56. The coRNA of claim 55, wherein the linker is single -stranded nucleic acid comprising from about from about 4 nucleotides to about 15 nucleotides.

57. The coRNA of any one of claims 47-56, wherein a loop portion (pin-loop) of the 5 ’-stem-loop comprises from 4, 5 or 6 nucleotides.

58. The coRNA of any one of claims 47-57, wherein a loop portion of the 5’-stem-loop comprises the nucleotide sequence GAAA or GAAAA.

59. The coRNA of any one of claims 47-58, wherein at least a portion of the 5’-stem-loop is within 45 nucleotides, counting from 5 ’-end, e.g., 5 ’-end of the conserved region of the parent coRNA.

60. The coRNA of any one of claims 47-59, wherein at least a portion of the stem of the 5 ’-stem- loop is within 25 nucleotides, counting from 5 ’-end, e.g., 5 ’-end of the conserved region of the parent coRNA.

61. The coRNA of any one of claims 47-60, wherein a stem portion of the 5 ’-stem-loop comprises a double -stranded region comprising from about 8 nucleotide base-pairs to about 25 nucleotide base-pairs.

62. The coRNA of any one of claims 47-61, wherein a stem portion of the 5’-stem-loop comprises a double-stranded region comprising about 9 nucleotide base-pairs, about 10 nucleotide base- pairs, about 11 nucleotide base-pairs, about 12 nucleotide base-pairs, about 13 nucleotide base- pairs, about 14 nucleotide base-pairs, about 15 base pairs nucleotide base-pairs, about 16 base pairs nucleotide base-pairs, about 17 base pairs nucleotide base-pairs, about 18 base pairs nucleotide base-pairs, about 19 base pairs nucleotide base-pairs, or about 20 base pairs nucleotide base -pairs.

63. The coRNA of any one of claims 47-62, wherein a stem portion of the 5 ’-stem-loop comprises a double-stranded region comprising about 9 nucleotide base-pairs, about 10 nucleotide base- pairs, about 11 nucleotide base-pairs, or about 12 nucleotide base-pairs.

64. The coRNA of any one of claims 47-63, wherein a stem portion of the 5 ’-stem-loop comprises a double -stranded region comprises 10 nucleotide base-pairs.

65. The coRNA of any one of claims 47-64, wherein the 5 ’-stem-loop comprises from about 20 nucleotides to about 65 nucleotides.

66. The coRNA of any one of claims 47-65, wherein the 5 ’-stem-loop comprises about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31,about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, or about 45 nucleotides.

67. The coRNA of any one of claims 47-66, wherein the 5 ’-stem -loop comprises 23, 24, 25, 26 or 27 nucleotides.

68. The coRNA of any one of claims 47-67, wherein the 5’-stem-loop comprises a nucleic acid modification, optionally selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof.

69. The coRNA of claim 68, wherein a stem (double -stranded) region of the 5 ’-stem-loop comprises a nucleic acid modification.

70. The coRNA of claim 68 or 69, wherein a loop region (pin-loop) of the 5 ’-stem-loop comprises a nucleic acid modification.

71. The coRNA of any one of claims 68-70, wherein said nucleic acid modification is a modified nucleotide selected from the group consisting of 2’-O-methyl (2’-OMe) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’-C-methylene (locked nucleic acid, LNA) nucleotides, 2’-O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl-carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene (Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-MOE) nucleotides, 2’-O- allyl nucleotides, 2’-C-allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-0-NMA) nucleotides, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O- aminopropyl (2'-O-AP) nucleotides, 2’-F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; a non-natural or modified nucleobase; or a combination thereof.

72. The coRNA of any one of claims 68-71, wherein a stem (double -stranded) region ofthe 5’-stem- loop comprises a 2’-O-methyl modified nucleotide.

73. The coRNA of any one of claims 68-71, wherein a stem (double -stranded) region ofthe 5’-stem- loop comprises a 2’-fluoro modified nucleotide.

74. The coRNA of any one of claims 68-71, wherein a nucleotide in a stem (double-stranded) region of the 5 ’-stem-loop comprises a non-natural or modified nucleobase.

75. The coRNA of any one of claims 68-74, wherein a stem (double -stranded) region of the 5 ’-stem- loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkage (e.g., a modified intemucleoside linkage selected independently from the group consisting of phosphorothioates (R. S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'- CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino ( — CH2-N(CH3)-O — CH2-), thiodiester ( — O —C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)2-O— and dialkylsiloxane), N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)- N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O- C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS- C5’), C3’-CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)-O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ internucleoside linkages, 2’->3’ internucleoside linkages, 3’->3’ internucleoside linkages, and 5’->5’ internucleoside linkages, optionally the modified internucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified internucleoside linkage is phosphorothioate (PS).

76. The ωRNA of claim 68-75, wherein a loop (single-stranded) region (pin-loop) of the 5’-stem- loop comprises a 2’-O-methyl modified nucleotide.

77. The ωRNA of any one of claims 68-75, wherein a loop (single-stranded) region (pin-loop) of the 5’-stem-loop comprises a 2’-fluoro modified nucleotide.

78. The ωRNA of any one of claims 68-77, wherein a nucleotide in a loop (single-stranded) (pin- loop) region of the 5’-stem-loop comprises a non-natural or modified nucleobase.

79. The ωRNA of any one of claims 68-78, wherein a loop (single-stranded) (pin-loop) region of the 5’-stem-loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified internucleoside linkage (e.g., a modified internucleoside linkage selected independently from the group consisting of phosphorothioates (R, S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino (—CH2-N(CH3)-O— CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)2-O— and dialkylsiloxane), N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’- N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’), C3’-CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)- O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ internucleoside linkages, 2’->3’ internucleoside linkages, 3’->3’ internucleoside linkages, and 5’->5’ internucleoside linkages, optionally themodified intemucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified intemucleoside linkage is phosphorothioate (PS).

80. The coRNA of any one of claims 47-79, wherein a stem (double -stranded) region of the 5 ’-stem- loop comprises a duplex stabilizing modification, optionally the duplex stabilizing modification is 2’-F nucleotide, 2’-OMe nucleotide, 2 ’-methoxy ethyl nucleotide, 2,6-diaminopurine nucleotide, 5-methyl cytidine, N4-ethyl cytidine, 5-propynyl cytidine, 5-propynyl uridine, 5- hydroxybutynl-2’ -deoxyuridine, 8-aza-7-deazaguanosine, a locked nucleic acid (LNA), and / or covalent cross-linking of two strands of the duplex.

81. The coRNA of any one of claims 47-80, wherein two nucleotides in the stem of the 5 ’-loop- stem are covalently linked to each other via a position other than 5 ’and 3’.

82. The coRNA of any one of claims 46-81, wherein the conserved region of the coRNA comprises at least two stem-loop structures and wherein said modification is in a stem-loop structure proximal to the 3’-end (3 ’-stem -loop) of the conserved region of the coRNA.

83. The coRNA of claim 82, wherein said modification comprises deletion of from about 1 to about 22 nucleotides from the 3 ’-stem -loop.

84. The coRNA of claim 82 or 83, wherein said modification comprises deletion of 2, 6, 8, 10, 12, 16, or 20 nucleotides from the 3 ’-stem-loop.

85. The coRNA of claim 84, wherein said modification comprises deletion of 2, 10, 12, or 16 nucleotides from the 3 ’-stem-loop.

86. The coRNA of claim 82-85, wherein said modification comprises deletion of 2, 10, 12 or 16 nucleotides from the 5 ’-stem-loop.

87. The coRNA of claim 86, wherein said modification comprises deletion of 2, 10, 12, or 16 nucleotides from the 3 ’-stem-loop.

88. The coRNA of any one of claims 82-87, wherein nucleotides at position 159-172, 160-171, 161- 170, 163-168, 158-173, 156-175 or 154-177 are deleted (counting from 5’-end, e.g., of the parent coRNA).

89. The coRNA of claim 88, wherein nucleotides at position 159-172, 163-168, 158-173, or 156- 175 are deleted (counting from 5 ’-end, e.g., of the parent coRNA).

90. The coRNA of any one of claims 82-89, wherein the deleted nucleotides in the 3 ’-stem-loop are replaced with a linker (e.g., a loop portion or pin-loop).

91. The coRNA of claim 90, wherein the linker is a single -stranded nucleic acid comprising from about from about 4 nucleotides to about 15 nucleotides.

92. The coRNA of any one of claims 82-91, wherein a loop portion of the 5’-stem-loop comprises from 4, 5 or 6 nucleotides.

93. The coRNA of any one of claims 82-92, wherein a loop portion of the 5 ’-stem-loop comprises the nucleotide sequence GAAA or GAAAA.

94. The coRNA of any one of claims 82-93, wherein at least a portion of the 3 ’-stem-loop is within 50 nucleotides, counting from 3 ’-end of the modified coRNA.

95. The coRNA of any one of claims 82-94, wherein at least a portion of the stem of the 5 ’-stem- loop is within 45 nucleotides, counting from 3 ’-end of the modified coRNA.

96. The coRNA of any one of claims 82-95, wherein a stem portion of the 3 ’-stem-loop comprises a double -stranded region comprising from about 3 nucleotide base-pairs to about 10 nucleotide base-pairs.

97. The coRNA of any one of claims 82-96, wherein a stem portion of the 3 ’-stem-loop comprises a double-stranded region comprising about 3 nucleotide base-pairs, about 4 nucleotide base- pairs, about 5 nucleotide base-pairs, about 6 nucleotide base-pairs, about 7 nucleotide base- pairs, or about 8 nucleotide base-pairs.

98. The coRNA of any one of claims 82-97, wherein a stem portion of the 3’-stem-loop comprises a double-stranded region comprising about 3 nucleotide base-pairs, about 4 nucleotide base- pairs, about 5 nucleotide base-pairs, or about 6 nucleotide base-pairs.

99. The coRNA of any one of claims 82-98, wherein a stem portion of the 5 ’-stem-loop comprises a double -stranded region comprises 3 or 4 nucleotide base-pairs.

100. The coRNA of any one of claims 82-99, wherein the 3 ’-stem -loop comprises from about 8 nucleotides to about 25 nucleotides.

101. The coRNA of any one of claims 78-100, wherein the 3’-stem-loop comprises about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides.

102. The coRNA of any one of claims 82-101, wherein the 3 ’-stem -loop comprises 9, 10, 11, 12, 13, 14 or 15 nucleotides.

103. The coRNA of any one of claims 78-102, wherein the 3’-stem-loop comprises a nucleic acid modification, optionally selected from the group consisting of nucleobase modifications, sugar modifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof.

104. The coRNA of claim 103, wherein a stem (double-stranded) region of the 3 ’-stem -loop comprises a nucleic acid modification.

105. The coRNA of claim 103 or 104, wherein a loop (single-stranded) region of the 3’-stem-loop comprises a nucleic acid modification.

106. The coRNA of any one of claims 103-105, wherein said nucleic acid modification is a modified nucleotide selected from the group consisting of 2’-O-methyl (2’-OMe) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’-C-methylene (locked nucleic acid, LNA) nucleotides, 2’-O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl-carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene(Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-M0E) nucleotides, 2’-O- allyl nucleotides, 2’-C-allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-0-NMA) nucleotides, a 2'-O-dimethylaminoethoxyethyl (2'-0-DMAE0E) nucleotides, 2'-O- aminopropyl (2'-O-AP) nucleotides, 2’-F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; a non-natural or modified nucleobase; or a combination thereof.

107. The coRNA of any one of claims 82-106, wherein a stem (double -stranded) region of the 3’- stem-loop comprises a 2’-O-methyl modified nucleotide.

108. The coRNA of any one of claims 82-107, wherein a stem (double -stranded) region of the 3’- stem-loop comprises a 2’-fluoro modified nucleotide.

109. The coRNA of any one of claims 82-108, wherein a nucleotide in a stem (double-stranded) region of the 5’-stem-loop comprises a non-natural or modified nucleobase.

110. The coRNA of any one of claims 82-109, wherein a stem (double -stranded) region of the 3’- stem-loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkage (e.g., a modified intemucleoside linkage selected independently from the group consisting of phosphorothioates ( / ?. S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'- CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino ( — CH2-N(CH3)-O — CH2-), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2-0 — and dialkylsiloxane), N,N'-dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)- N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CEE-O-S'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O- C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS- C5’), C3’-CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)-O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ intemucleoside linkages, 2’->3’ intemucleoside linkages, 3’->3’ intemucleoside linkages, and 5 ’->5’ intemucleoside linkages, optionally the modified intemucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified intemucleoside linkage is phosphorothioate (PS).

111. The coRNA of any one of claims 82-110, wherein a loop (single -stranded) region of the 5’- stem-loop comprises a 2’-O-methyl modified nucleotide.

112. The coRNA of any one of claims 82-111, wherein a loop (single -stranded) region of the 5’- stem-loop comprises a 2’-fluoro modified nucleotide.

113. The coRNA of any one of claims 82-112, wherein a nucleotide in a loop (single-stranded) region of the 3 ’-stem-loop comprises a non-natural or modified nucleobase.

114. The coRNA of any one of claims 82-113, wherein a loop (single -stranded) region of the 5’- stem-loop comprises at least one (e.g., 1, 2, 3, 4, 5, or more) modified intemucleoside linkage (e.g., a modified intemucleoside linkage selected independently from the group consisting of phosphorothioates (R. S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'- CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino ( — CH2-N(CH3)-O — CH2-), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2-0 — and dialkylsiloxane), N,N'-dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)- N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O- C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS- C5’), C3’-CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)-O-5', 3'-NHP(O)(OCH3)-O-5’), 2’->5’ intemucleoside linkages, 2’->3’ intemucleoside linkages, 3’->3’ intemucleoside linkages, and 5 ’->5’ intemucleoside linkages, optionally the modified intemucleoside linkage is phosphorothioate, imidp or MMI, more preferably the modified intemucleoside linkage is phosphorothioate (PS).

115. The coRNA of any one of claims 82-114, wherein two nucleotides in the stem of the 5 ’-loop- stem are covalently linked to each other via a position other than 5 ’and 3’.

116. The coRNA of any one of claims 82-115, wherein two nucleotides in the stem of the 5’-loop- stem are covalently linked to each other via a position other than 5 ’and 3’.

117. The coRNA of any one of claims 46-116, wherein the conserved region of the coRNA comprises at least three stem -loop structures.

118. The coRNA of any one of claims 46-117, wherein at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) nucleotide in a loop region of a stem -loop forms a base-pair with a nucleotide outside said stem- loop.

119. The coRNA of claim 118, wherein at least one (e.g., one or both) member of the base-pair comprises a modification capable of enhancing base-pairing.

120. The coRNA of claim 119, wherein the base-pair nucleotide in the loop comprises the modification capable of enhancing base-pairing.

121. The coRNA of claim 119 or 120, wherein the base-pair nucleotide outside said stem-loop comprises the modification capable of enhancing base-pairing.

122. The coRNA of any one of claims 119-121, wherein said modification capable of enhancing base-pairing is 2’-F modified nucleotide, 2’-0Me modified nucleotide, 2 ’-methoxy ethyl modified nucleotide, 2,6-diaminopurine nucleotide, 5-methyl cytidine, N4-ethyl cytidine, 5- propynyl cytidine, 5-propynyl uridine, 5 -hydroxybutynl-2’ -deoxyuridine, 8-aza-7- deazaguanosine, or a locked nucleic acid (LNA).

123. The coRNA of any one of claims 119-122, wherein said modification capable of enhancing base-pairing is in at an internal (i.e., not a 5’-stem-loop or 3’-stem-loop) stem-loop (internal- hairpin) of the coRNA.

124. The coRNA of any one of claims 119-123, wherein said modification capable of enhancing base-pairing is in a loop (single-stranded) region (pin-loop) of an internal (i.e., not a 5’-stem- loop or 3 ’-stem -loop) stem -loop (internal-hairpin) of the coRNA.

125. The coRNA of any one of claims 46-124, wherein the coRNA (e.g., a conserved region) is from about 50 to about 90 nucleotides shorter than the parent coRNA.

126. The coRNA of any one of claims 46-125, wherein the coRNA (e.g., a conserved region) is from about 55 to about 85 nucleotides shorter than the parent coRNA.

127. The coRNA of any one of claims 46-126, wherein the coRNA (e.g., a conserved region) is about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, or about 80 nucleotides shorter than the parent coRNA.

128. The coRNA of any one of claims 46-127, wherein the coRNA (e.g., a conserved region) is from about 130 nucleotides to about 190 nucleotides in length.

129. The coRNA of any one of claims 46-128, wherein the coRNA (e.g., a conserved region) is from about 135 nucleotides to about 180 nucleotides in length.

130. The coRNA of any one of claims 46-129, wherein the coRNA (e.g., a conserved region) is from about 140 nucleotides to about 170 nucleotides in length.

131. The coRNA of any one of claims 46-130, wherein the coRNA (e.g., a conserved region) is from about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 1454, about 155, about 156, about 157, about 158, about 159, or about 160 nucleotides in length.

132. The coRNA of any one of claims 46-131, wherein the coRNA comprises a 5’ end modification.

133. The coRNA of any one of claims 46-132, wherein the coRNA comprises a 3’ end modification.

134. The coRNA of any one of claims 46-133, wherein the coRNA comprises a 5’ end modification and a 3’ end modification.

135. The coRNA of any one of claims 132-134, wherein the 5’ and / or 3’ end modification comprises a protective end modification.

136. The coRNA of claim 135, wherein the protective end modification is a modified nucleotide selected from 2’-O-methyl (2’-OMe) modified nucleotide, 2’-O-(2-methoxyethyl) (2’- 0-moe)modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, a 3 ’->3’ linkage between nucleotides, an inverted abasic modified nucleotide, or combinations thereof.

137. The coRNA of any one of claims 132-136, wherein the 3’ end modification comprises any one of: i. a modification of any one or more of the last 7, 6, 5, 4, 3, 2, or 1 nucleotides; ii. one modified nucleotide; iii. two modified nucleotides; iv. three modified nucleotides; v. four modified nucleotides; vi. five modified nucleotides; vii. six modified nucleotides; or viii. seven modified nucleotides.

138. The coRNA of any one of claims 132-137, wherein the 3’ end modification i. a modified intemucleoside linkage (e.g., PS, imidp or MMI linkage) between nucleotides; ii. a 2’-OMe nucleotide; iii. a 2’-0-M0E nucleotide; iv. a 2’ -F nucleotide; v. a LNA nucleotide vi. a 3’->3’ linkage between nucleotides; vii. an inverted abasic nucleotide; and viii. a combination of one or more of (i.) - (vii.).

139. The coRNA of any one of claims 132-138, wherein the 3’ end modification comprises one or more of: i. a modified intemucleoside linkage (e.g., PS, imidp or MMI linkage) between the last and second to last nucleotides; ii. a modified intemucleoside linkage (e.g., PS, imidp or MMI linkage) between the last and second to last nucleotide and a PS linkage between the second and third to last nucleotides; iii. modified intemucleoside linkages (e.g., PS and / or MMI linkage) between any one or more of the last four nucleotides; iv. modified intemucleoside linkages (e.g., PS and / or MMI linkage) between any one or more of the last five nucleotides; or v. modified intemucleoside linkages (e.g., PS and / or MMI linkage) between any one or more of the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

140. The coRNA of any one of claims 132-139, wherein the 3’ end modification comprises at least one 2’-F nucleotide, 2’-OMe nucleotide, 2’-O-MOE nucleotide, inverted abasic nucleotide or a 3 ’->3’ intemucleoside linkage.

141. The coRNA of any one of claims 132-140, wherein the 3’ end modification comprises: i. a modification of one or more of the last 1-7 nucleotides, wherein the modification is a modified intemucleoside linkages (e.g., PS and / or MMI linkage), inverted abasic nucleotide, a 3’->3’ intemucleoside linkage, 2’-OMe, 2’-O-MOE, 2’-F, LNA, or a combination thereof; ii. a modification to the last nucleotide with 2’-OMe, 2’-O-MOE, 2’-F, LNA, or combinations thereof, and an optional one or two modified intemucleoside linkages (e.g., PS and / or MMI linkage) to the next nucleotide; iii. a modification to the last and / or second to last nucleotide with 2’-OMe, 2’-O-MOE, 2’- F, LNA, or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage); iv. a modification to the last, second to last, and / or third to last nucleotides with 2’-OMe, 2’-O-moe, 2’-F, or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage); v. a modification to the last, second to last, third to last, and / or fourth to last nucleotides with 2’-OMe, 2’-0-M0E, 2’-F, LNA or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage); or vi. a modification to the last, second to last, third to last, fourth to last, and / or fifth to last nucleotides with 2’-OMe, 2’-0-M0E, 2’-F, LNA, or combinations thereof, and optionally one or more modified intemucleoside linkages (e.g., PS and / or MMI linkage).

142. The coRNA of any one of claims 132-141, wherein the 3’ end modification comprises any one or more of:(i) a 2’-OMe modified nucleotide at the last position, three consecutive 2’-0-M0E modified nucleotides immediately 5’ to the 2’-OMe modified nucleotide, and three consecutive PS linkages between the last three nucleotides;(ii) five consecutive 2’-OMe modified nucleotides from the 3’ end of the 3’ terminus, and three PS linkages between the last three nucleotides;(iii) an inverted abasic modified nucleotide at the last position;(iv) an inverted abasic modified nucleotide at the last position, and three consecutive 2’- OMe modified nucleotides at the last three positions(v) 15 consecutive 2’-OMe modified nucleotides from the 3’ end, five consecutive 2’-F modified nucleotides immediately 5’ to the 2’-OMe modified nucleotides, and three PS linkages between the last three nucleotides;(vi) alternating 2’-0Me modified nucleotides and 2’-F modified nucleotides at the last 20 nucleotides, and three PS linkages between the last three nucleotides;(vii) two or three consecutive 2’-OMe modified nucleotides, and three PS linkages between the last three nucleotides;(viii) one PS linkage between the last and next to last nucleotides; and(ix) 15 or 20 consecutive 2’-OMe modified nucleotides, and three PS linkages between the last three nucleotides.

143. The coRNA of any one of claims 132-142, wherein the 5’ end modification comprises any one or more of: i. one modified nucleotide; ii. two modified nucleotides; iii. three modified nucleotides; iv. four modified nucleotides; v. five modified nucleotides; vi. six modified nucleotides; and vii. seven modified nucleotides.

144. The coRNA of any one claims 132-143, wherein the 5’ end modification comprises any one or more of: a modification of between 1 and 7, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2 nucleotides.

145. The coRNA of any one of claims 132-144, wherein the 5’ end modification comprises any one or more of: i. a modified intemucleoside linkage (e.g., a phosphorothioate and / or MMI linkage) between nucleotides; ii. a 2’-OMe nucleotide; iii. a 2’-O-MOE nucleotide; iv. a 2’ -F nucleotide; v. a LNA; vi. a 3 ’->3’ linkage; vii. an inverted abasic modified nucleotide; viii. a deoxyribonucleotide; ix. an inosine; and x. combinations of one or more of (i.) - (ix.).

146. The coRNA of any one of claims 132-145, wherein the 5’ end modification comprises: i. 1, 2, 3, 4, 5, 6, and / or 7 modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between nucleotides; or ii. about 1-2, 1-3, 1-4, 1-5, 1-6, or 1-7 modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between nucleotides.

147. The coRNA of any one claims 132-146, wherein the 5’ end modification comprises: i. there is one modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkage, and the linkage is between nucleotides 1 and 2; ii. there are two modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages, and the linkages are between nucleotides 1 and 2, and 2 and 3; iii. there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, and 3 and 4; iv. there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, and 4 and 5; v. there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6; vi. there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, and 6 and 7; or vii. there are modified intemucleoside (e.g., a phosphorothioate and / or MMI) linkages between any one or more of nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, and 7 and 8.

148. The coRNA of any one of claims 132-147, wherein the 5’ end modification comprises at least one 2’-0Me, 2’-0-M0E, inverted abasic, or 2’-F modified nucleotide.

149. The coRNA of any one of claims 46-148, wherein the coRNA further comprises a guide region capable of hybridizing with a target nucleic acid.

150. The coRNA of any one of claims 46-149, wherein the coRNA is hybridized with a second nucleic acid strand.

151. The coRNA of claim 150, wherein the second strand comprises a guide region comprising a sequence capable of hybridizing with a target nucleic acid.

152. The coRNA of any one of claims 149-151, wherein the guide region is from about 15 to about 30 nucleotides in length.

153. The coRNA of any one of claims 149-152, wherein the guide region is from about 18 to about 25 nucleotides in length.

154. The coRNA of any one of claims 149-153, wherein the guide region is from 18, 19, 20, 21, 22 or 23 nucleotides in length.

155. The coRNA of any one of claims 149-154, wherein the guide region is at 5’-end of the coRNA.

156. The coRNA of any one of claims 149-154, wherein the guide region is at 3’-end of the coRNA.

157. The coRNA of any one of claims 149-156, wherein the guide region comprises a nucleic acid modification, optionally selected from the group consisting of nucleobase modifications, sugarmodifications, inter-sugar linkage modifications (modified intemucleoside linkages), conjugates (e.g.., ligands), and any combinations thereof.

158. The coRNA of claim 157, wherein said nucleic acid modification is a modified nucleotide selected from the group consisting of 2’-O-methyl (2’-0Me) nucleotides, 2’-fluoro (2’-F) nucleotides, bridged nucleic acid (BNA) nucleotides (e.g., 2’-O,4’-C-methylene (locked nucleic acid, LNA) nucleotides, 2’-O,4’-C-ethylene (locked nucleic acid, ENA) nucleotides, 5 ’-methyl -BNA, cEt BNA, cMOE BNA, oxy amino BNA and vinyl-carbo BNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA) nucleotides, cyclohexene (Cyclohexene nucleic acid, CeNA) nucleotides, 2 ’-methoxy ethyl (2’-M0E) nucleotides, 2’-O- allyl nucleotides, 2’-C-allyl ribose nucleotides, 2'-O-N-methylacetamido (2'-0-NMA) nucleotides, a 2'-O-dimethylaminoethoxyethyl (2'-0-DMAE0E) nucleotides, 2'-O- aminopropyl (2'-O-AP) nucleotides, 2’-F arabinose (2'-ara-F) nucleotides, threose (Threose nucleic acid, TNA) nucleotides, and acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA)); a modified intemucleoside linkage; or a non-natural or modified nucleobase.

159. The coRNA of any one of claims 149-156, wherein the guide region comprises: i. a modification of one or more of nucleotides 1-7, wherein the modification is a modified intemucleoside (e.g., phosphorothioate and / or MMI) linkage, 3 ’->3’ intemucleoside linkage, inverted abasic nucleotide, 2’-0Me, 2’-0-M0E, 2’-F, 2’-H (a deoxyribonucleotide), an inosine, and / or combinations thereof; ii. a modification to the first nucleotide of the guide region with 2’-0Me, 2’-0-M0E, 2’-F, 2’-H, an inosine, or combinations thereof, and an optional modified intemucleoside (e.g., phosphorothioate and / or MMI) linkage to the next nucleotide; iii. a modification to the first and / or second nucleotide of the guide region with 2’-0Me, 2’- O-MOE, 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages; iv. a modification to the first, second, and / or third nucleotides of the guide region with 2’- OMe, 2’-0-M0E 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages; v. a modification to the first, second, third, and / or fourth nucleotides of the guide region with 2’-0Me, 2’-0-M0E, 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages; or vi. a modification to the first, second, third, fourth, and / or fifth nucleotides of the guide region with 2’-0Me, 2’-0-M0E, 2’-F, 2’-H, an inosine, or combinations thereof, and optionally one or more modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages.

160. The coRNA of any one of claims 149-159, wherein the coRNA comprises any one or more of: i. 2’-OMe nucleotides at nucleotides 1, 2, and 3 of the guide region; ii. 2’-OMe nucleotides at nucleotides 1, 2, and 3 of the guide region and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; iii. 2’-OMe nucleotides at nucleotides 1, 2, 3, 4, and 5 of the guide region; iv. 2’-OMe nucleotides at nucleotides 1, 2, 3, 4, and 5 of the guide region and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the guide region; v. 2’-0-M0E modified nucleotides at nucleotides 1, 2, and 3 of the guide region; vi. 2’-0-M0E modified nucleotides at nucleotides 1, 2, and 3 of the guide region and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; vii. an inverted abasic nucleotide at nucleotide 1 of the guide region; viii. an inverted abasic nucleotide at nucleotide 1 of the guide region and 2’-0Me nucleotides at nucleotides 1, 2, and 3 of the guide region; and ix. an inverted abasic nucleotide at nucleotide 1 of the guide region, 2’-0Me nucleotides at nucleotides 1, 2, and 3 of the guide region, and modified intemucleoside (e.g., phosphorothioate and / or MMI) linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the variable region.

161. The coRNA of any one of claims 46-160, wherein the coRNA comprises one of more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more including all) modified pyrimidine-A dinucleotide (modified YA site).

162. The coRNA of any one of claims 46-161, wherein the coRNA comprises at least one modified YA site in the guide region.

163. The coRNA of any one claims 46-162, wherein the coRNA comprises at least one modified YA site in a conserved region of the coRNA.

164. The coRNA of any one of claims 46-163, wherein the modified YA site comprises a 2 ’-fluoro nucleotide, optionally the pyrimidine of the YA site; a 2’-H nucleotide, optionally the pyrimidine of the YA site; a 2’-OMe nucleotide, optionally the pyrimidine of the YA site; a PS linkage between the nucleotides; or a combination thereof.

165. The coRNA of any one of claims 46-164, wherein the coRNA comprises: i. a modified YA site at 1, 2, 3, 4, or 5 guide region YA sites; ii. a modified YA site at 1, 2, 3, 4, or 5 guide region YA sites, wherein the modification of at least one guide region YA site is different from any 5’ end modification of the coRNA; iii. a modified YA site at one or more guide region YA sites that are at or after nucleotide 8 from the 5 ’ end of the 5 ’ terminus;iv. a modified YA site at one or more guide region YA sites that are within nucleotides 5- end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5’ end of the 5’ terminus; v. a modified YA site at one or more guide region YA sites that are within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3’ terminal nucleotide of the guide region; vi. a modified YA site at a guide region YA site other than a 5’ end modification; or vii. a modified YA site at a guide region YA site, wherein the modification of the guide region YA site comprises a modification at least one nucleotide located 5’ of the guide region YA site does not comprise.

166. The coRNA of any one of claims 46-165, wherein the coRNA comprises: i. a modified YA site at two or more guide region YA sites that are at or after nucleotide 8 from the 5 ’-end; ii. a modified YA site at two or more guide region YA sites that are within nucleotides 5- end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5’ end; iii. a modified YA site at two or more guide region YA sites that are within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3’ terminal nucleotide of the guide region; iv. a modified YA site at two or more guide region YA sites other than a 5’ end modification; or v. a modified YA site at a two or more guide region YA sites, wherein the modifications of the guide region YA sites comprise a modification at least one nucleotide located 5’ of the guide region YA site does not comprise.

167. The coRNA of any one of claims 46-166, wherein the coRNA comprises: i. a modified YA site at three or more guide region YA sites that are at or after nucleotide 8 from the 5 ’ end of the 5 ’ terminus; ii. a modified YA site at three or more guide region YA sites that are within nucleotides 5- end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5’ end of the 5’ terminus; iii. a modified YA site at three or more guide region YA sites that are within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3’ terminal nucleotide of the guide region; iv. a modified YA site at three or more guide region YA sites other than a 5 ’ end modification; or v. a modified YA site at a three or more guide region YA sites, wherein the modifications of the guide region YA sites comprise a modification at least one nucleotide located 5’ of the guide region YA site does not comprise.

168. The coRNA of any one of claims 46-167, wherein the coRNA retains at least 75% of the activity of the parent coRNA.

169. The coRNA of any one of claims 46-168, wherein the nuclease is a transposon family nuclease.

170. The coRNA of claim 169, wherein nuclease is a IS200 / IS605 transposon family encoded nuclease or a homolog or ortholog thereof.

171. The coRNA of claim 170, wherein the nuclease is ISC (insertion sequences Cas9-like), TnpB, or a homolog or ortholog thereof.

172. The coRNA of claim 171, wherein the nuclease is IscB nuclease, IsrB nuclease, TnpB nuclease, or a homolog or ortholog thereof, optionally, the nuclease is IscB nuclease or a homolog or ortholog thereof.

173. The coRNA of any one of claims 46-172, wherein the nuclease is modified nuclease.

174. The coRNA of any one of claims 46-173, wherein the coRNA lacks from 1 to about 20 nucleotides present at 3 ’-end of the conserved portion of the parent coRNA.

175. A vector comprising the coRNA of any one of claims 46-174.

176. A composition comprising a coRNA of any one of claims 46-174 or a polynucleotide encoding the same.

177. The composition of claim 176, wherein the composition is in form of a lipid nanoparticles (LNP) or a protein nanoparticles (PNP).

178. The composition of any one of claims 176-177, wherein the composition further comprises a nuclease or a polynucleotide encoding the nuclease.

179. The composition of claim 178, wherein the nuclease is a transposon family nuclease.

180. The composition of claim 178-179, wherein nuclease is a IS200 / IS605 transposon family encoded nuclease or a homolog or ortholog thereof.

181. The composition of claim 180, wherein the nuclease is ISC, TnpB, or a homolog or ortholog thereof.

182. The composition of claim 181, wherein the nuclease is IscB, IsrB, TnpB nuclease, or a homolog or ortholog thereof, optionally, the nuclease is IscB or a homolog or ortholog thereof.

183. The composition of any one of claims 178-182, wherein the nuclease is a modified nuclease.

184. A pharmaceutical composition comprising an IscB protein of any one of claims 1-31, a fusion protein of any one of claims 32-45, a coRNA of any one of claims 46-174, and / or a composition of any one of claims 176-183.

185. A DNA and RNA targeting system comprising: (i) a protein effector polypeptide or a polynucleotide encoding the protein effector polypeptide, optionally, the protein effector polypeptide has nuclease (e.g., endonuclease activity); and (ii) a coRNA of any one of claims 46-174 or a polynucleotide encoding the coRNA.

186. A DNA and RNA targeting composition comprising: (i) a protein effector polypeptide or a polynucleotide encoding the protein effector polypeptide, optionally, the protein effector polypeptide has nuclease (e.g., endonuclease activity); and (ii) a coRNA of any one of claims 46-174 or a polynucleotide encoding the coRNA.

187. The DNA and RNA targeting system of claim 185 or the DNA and RNA targeting composition of claim 186, wherein the nuclease is a transposon family nuclease.

188. The DNA and RNA targeting system or composition of claim 187, wherein nuclease is a IS200 / IS605 transposon family encoded nuclease or a homolog or ortholog thereof.

189. The DNA and RNA targeting system or composition of claim 188, wherein the nuclease is ISC, TnpB, or a homolog or ortholog thereof.

190. The DNA and RNA targeting system or composition of claim 189, wherein the nuclease is IscB, IsrB, TnpB nuclease, or a homolog or ortholog thereof, optionally, the nuclease is IscB or a homolog or ortholog thereof.

191. The DNA and RNA targeting system or composition of any one of claims 185-190, wherein the nuclease is a modified nuclease.

192. The DNA and RNA targeting system or composition of any one of claims 185-191, wherein the protein effector polypeptide comprises a RuvC domain, and / or an HNH domain.

193. The DNA and RNA targeting system or composition of any one of claims 185-192, wherein the protein effector polypeptide comprises: (i) a split Ruv-C nuclease domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains; and / or (ii) an HNH domain.

194. The DNA and RNA targeting system or composition of any one of claims 185-193, wherein the protein effector polypeptide comprises a PLMP domain and optionally a conserved C- terminal Y domain.

195. The DNA and RNA targeting system or composition of any one of claims 192-194, wherein the HNH domain is located in the RuvC domain (e.g., the HNH domain is located between RuvC-II and RuvC-III subdomains).

196. The DNA and RNA targeting system or composition of any one of claims 192-194, wherein the protein effector polypeptide comprises a HNH domain but no RuvC domain.

197. The DNA and RNA targeting system or composition of any one of claims 192-194, wherein the protein effector polypeptide comprises a RuvC (e.g., split RuvC) domain but no HNH domain.

198. The DNA and RNA targeting system or composition of any one of claims 192-197, wherein the protein effector polypeptide further comprises a REC domain.

199. The DNA and RNA targeting system or composition of claim 198, wherein the REC domain is downstream from the RuvC or HNH domain.

200. The DNA and RNA targeting system or composition of claim 198, wherein the REC domain is upstream from the RuvC or HNH domain.

201. The DNA and RNA targeting system or composition of any one of claims 185-200, wherein the protein effector polypeptide comprises from about 170 to about 1000 amino acids.

202. The DNA and RNA targeting system or composition of any one of claims 185-201, wherein the coRNA comprises a guide sequence comprising a nucleotide sequence substantially complementary to at least a portion of a target nucleic acid (target sequence)203. The DNA and RNA targeting system or composition of claim 202, wherein the target nucleic acid comprises a target adjacent motif (TAM) sequence 3’ of the target sequence.

204. The DNA and RNA targeting system of claim 203, wherein the TAM comprises the sequence NAC, where N is A, C, G, or T.

205. The DNA and RNA targeting system or composition of any one of claims 202-204, wherein the target nucleic acid is DNA.

206. The DNA and RNA targeting system or composition of any one of claims 185-205 further comprising a functional domain associated with the protein effector polypeptide.

207. The DNA and RNA targeting system or composition of claim 206, wherein the functional domain has transposase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double- strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, nucleic acid binding activity, detectable activity, or any combination thereof.

208. The DNA and RNA targeting system or composition of any one of claims 185-207, further comprising a serine or tyrosine recombinase.

209. The DNA and RNA targeting system or composition of any one of claims 185-208, further comprising a homologous recombination donor template comprising a donor sequence for insertion into a target polynucleotide.

210. An engineered cell comprising a DNA and RNA targeting system or composition of any one of claims 185-209.

211. A complex comprising the fusion protein of any one of claims 32-45, and a guide RNA bound to the IscB protein.

212. A complex comprising the fusion protein of any one of claims 32-45, and a guide RNA comprising the coRNA of any one of claims 46-174 bound to the IscB protein.

213. The complex of claim 211 or 212, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

214. The complex of any one of claims 211-213, wherein the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.

215. The complex of any one of claims 211-214, wherein the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence.

216. The complex of any one of claims 211-215, wherein the target sequence is a DNA sequence.

217. The complex of claim 216, wherein the target sequence is a sequence in the genome of a mammal.

218. The complex of claim 217, wherein the target sequence is a sequence in the genome of a human.

219. The complex of any one of claims 211-218, wherein the target sequence comprises a sequence associated with a disease or disorder.

220. The complex of claim 219, wherein the target sequence comprises a point mutation associated with a disease or disorder.

221. The complex of claim 220, wherein the complex edits a point mutation in the target sequence.

222. The complex of claim 221, wherein the point mutation is located between about 10 to about 20 nucleotides upstream of the TAM in the target sequence.

223. The complex of claim 221 or 222, wherein the target sequence comprises a T to C point mutation.

224. The complex of claim 223, wherein the complex deaminates the target C point mutation, and wherein the deamination results in a sequence that is not associated with a disease or disorder.

225. The complex of claim 224, wherein the target C point mutation is present in the DNA strand that is not complementary to the guide RNA.

226. The complex of claim 221 or 222, wherein the target sequence comprises a G to A point mutation.

227. The complex of claim 226, wherein the complex deaminates the target A point mutation, and wherein the deamination results in a sequence that is not associated with a disease or disorder.

228. The complex of claim 227, wherein the target A point mutation is present in the DNA strand that is not complementary to the guide RNA.

229. The complex of any one of claims 211-228, wherein the complex exhibits increased deamination efficiency of a point mutation in a target sequence that does not comprise the canonical TAM (5’-ATAAA-3’) at its 3' end as compared to the deamination efficiency of a complex comprising IscB protein as provided by SEQ ID NO: 2.

230. The complex of claim 229, wherein the complex exhibits increased deamination efficiency of a point mutation in a target sequence having a 3 ' end that is not directly adjacent to the canonical TAM sequence (5’-ATAAA-3’) that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the deamination efficiency ofcomplex comprising the IscB protein as provided by SEQ ID NO: 2 on the same target sequence.

231. The complex of any one of claims 224-230, wherein a deamination activity is measured using a deamination assay, PCR, or sequencing.

232. The complex of any one of claims 211-231, wherein the complex produces fewer indels in a target sequence that does not comprise the canonical TAM (5’-ATAAA-3’) at its 3' end as compared to the amount of indels produced by a complex comprising IscB protein as provided by SEQ ID NO: 2.

233. The complex of claim 232, wherein the complex produces fewer indels in a target sequence having a 3' end that is not directly adjacent to the canonical TAM sequence (5’-ATAAA-3’) that is at least 2-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 10-fold, at least 50- fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000- fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold lower as compared to the amount of indels produced by a complex comprising IscB protein as provided by SEQ ID NO: 2 on the same target sequence.

234. The complex of any one of claims 232-233, wherein indels are measured using high-throughput sequencing.

235. The complex of any one of claims 211 -234, wherein the complex exhibits a decreased off-target activity as compared to the off-target activity of a complex comprising IscB protein as provided by SEQ ID NO: 2.

236. The complex of claim 235, wherein the off-target activity of the complex is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold decreased as compared to the off-target activity of a complex comprising IscB protein as provided by SEQ ID NO: 2.

237. The complex of any one of claims 211-236, wherein the target sequence is in the genome of an organism.

238. The complex of claim 237, wherein the organism is a prokaryote.

239. The complex of claim 237, wherein the prokaryote is a bacterium.

240. The complex of claim 237, wherein the organism is a eukaryote.

241. The complex of claim 237, wherein the organism is a plant or fungus.

242. The complex of claim 237, wherein the organism is a vertebrate.

243. The complex of claim 242, wherein the vertebrate is a mammal.

244. The complex of claim 243, wherein the mammal is a human.

245. The complex of claim 237, wherein the organism is a cell.

246. The complex of claim 245, wherein the cell is a human cell.

247. A method comprising contacting a nucleic acid with the fusion protein of any one of claims 32- 45, and with a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

248. A method comprising contacting a cell with the fusion protein of any one of claims 32-45, and with a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

249. A method comprising contacting a nucleic acid with the fusion protein of any one of claims 32- 45, and with a guide RNA comprising the coRNA of any one of claims 46-174.

250. A method comprising contacting a cell with the fusion protein of any one of claims 32-45, and with a guide RNA comprising the coRNA of any one of claims 46-174.

251. A method comprising contacting a nucleic acid with the complex of any one of claims 211 -246.

252. A method comprising contacting a cell with the complex of any one of claims 211-246.

253. The method of any one of claims 247-252, wherein the contacting is performed in vitro.

254. The method of any one of claims 247-253, wherein the contacting is performed in vivo.

255. A method comprising administering to a subject the fusion protein of any one of claims 32-45, and a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

256. A method comprising administering to a subject the fusion protein of any one of claims 32-45, and a guide RNA comprising the coRNA of any one of claims 46-174.

257. A method comprising administering to a subject the complex of any one of claims 211-246 or the DNA and RNA targeting system or composition of any one of claims 185-209.

258. The method of any one of claims 247-257, wherein the target sequence of the nucleic acid is a DNA sequence.

259. The method of any one of claims 247-258, wherein the 3' end of the target sequence is not immediately adjacent to the canonical TAM sequence (5’-ATAAA-3’j.

260. The method of any one of claims 247-259 wherein the target sequence comprises a sequence associated with a disease or disorder.

261. The method of claim 260, wherein the target DNA sequence comprises a point mutation associated with a disease or disorder.

262. The method of claim 261, wherein the activity of the fusion protein, or the activity of the complex, results in a correction of the point mutation.

263. The method of any one of claims 259-262, wherein the target DNA sequence comprises a T to C point mutation associated with a disease or disorder, and wherein the deamination of the mutant C base results in a sequence that is not associated with a disease or disorder.

264. The method of claim 263, wherein the target DNA sequence encodes a protein and wherein the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon.

265. The method of claim 263 or 264, wherein the deamination of the mutant C results in a change of the amino acid encoded by the mutant codon.

266. The method of claim 263, wherein the deamination of the mutant C results in the codon encoding the wild-type amino acid.

267. The method of any one of claims 261-262, wherein the target DNA sequence comprises a G to A point mutation associated with a disease or disorder, and wherein the deamination of the mutant A base results in a sequence that is not associated with a disease or disorder.

268. The method of claim 267, wherein the target DNA sequence encodes a protein and wherein the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon.

269. The method of claim 268, wherein the deamination of the mutant A results in a change of the amino acid encoded by the mutant codon.

270. The method of claim 267, wherein the deamination of the mutant A results in the codon encoding the wild-type amino acid.

271. The method of any one of claims 247-270, wherein the contacting is in vivo in a subject.

272. The method of claim 271, wherein the subject has or has been diagnosed with a disease or disorder.

273. The method of claim 271 or 272, wherein the disease or disorder is a proliferative disease, a genetic disease, a neoplastic disease, a metabolic disease, or a lysosomal storage disease.

274. A kit comprising a nucleic acid construct, comprising:(a) a nucleic acid sequence encoding the fusion protein of any one of claims 32-45; and (b) a heterologous promoter that drives expression of the sequence of (a).

275. A kit comprising a nucleic acid construct, comprising:(a) a nucleic acid sequence encoding the complex of any one of claims 211-246; and (b) a heterologous promoter that drives expression of the sequence of (a).

276. The kit of 274 or 275, further comprising an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.

277. The kit of any one of claims 274-276, further comprising an expression construct encoding a guide RNA comprising the coRNA of any one of claims 46-174.

278. A polynucleotide encoding the fusion protein of any one of claims 32-45 or the complex of any one of claims 211-246 or the DNA and RNA targeting system or composition of any one of claims 185-209.

279. A vector comprising a polynucleotide of claim 278.

280. The vector of claim 279, wherein the vector comprises a heterologous promoter driving expression of the polynucleotide encoding the fusion protein or the polynucleotide encoding the complex.

281. A method comprising contacting a cell with the vector of claim 279 or 280.

282. The method of claim 281, wherein the vector is transfected into the cell.

283. The method of claim 282, wherein the vector is transfected into the cell using electroporation, heat shock, or a composition comprising a cationic lipid.

284. A cell comprising the fusion protein of any one of claims 32-45, or a nucleic acid molecule encoding the fusion protein of any one of claims 32-45.

285. A cell comprising the complex of any one of claims 211-246, or a nucleic acid molecule encoding the complex of any one of claims 211-246.

286. A cell comprising the vector of claim 279 or 280.

287. A DNA and RNA targeting composition comprising an IscB protein of any one of claims 1- 31 and a guide nucleic acid capable of binding with the IscB protein, wherein the guide nucleic acid comprises a guide sequence complementary to target sequence and capable of directing the IscB protein to a target polynucleotide.

288. A DNA and RNA targeting composition comprising an IscB protein of any one of claims 1- 31 and a guide nucleic acid comprising the coRNA of any one of claims 46-174.

289. The composition of claim 287 or 288, wherein the guide is from about 10 nucleotides to about 150 nucleotides in length, preferably 12 to 50 nt, more preferably 15 and 45 nt in length.

290. The composition of any one of claims 287-289, wherein the target sequence comprises a target adjacent motif (TAM) sequence 3’ of the target sequence.

291. The composition of any of claims 287-290, wherein the target polynucleotide is DNA.

292. The composition of any of claims 287-291, wherein the guide nucleic acid further comprises an aptamer.

293. The composition of any of claims 287-292, wherein the guide nucleic acid further comprises an extension to add an RNA template.

294. The composition of any of claims 287-293, further comprising a functional domain associated with the IscB protein.

295. The composition of claim 294, wherein the functional domain has transposase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, nucleic acid binding activity, detectable activity, or any combination thereof.

296. The composition of claim 294 or 295, wherein the functional domain is covalently linked with the IscB protein.

297. The composition of claim 296, wherein the IscB is fusion protein of any one of claims 32-45298. The composition of any one of claims 287-297, further comprising a serine or tyrosine recombinase.

299. The composition of any one of claims 287-298, further comprising a homologous recombination donor template comprising a donor sequence for insertion into a target polynucleotide.

300. The composition of any one of claims 287-299, wherein a nuclease domain of the IscB protein is catalytically inactive.

301. The composition of any one of claims 287-300, wherein the nuclease domain has nickase activity or is engineered to have nickase activity.

302. The composition of any one of claims 287-301, wherein the target sequence comprises a TAM sequence of ATR, where R is purine (e.g., G or A).

303. An engineered cell comprising the composition of any one of claims 287-302.

304. A method of modifying a target sequence in a cell, the method comprising administering a composition of any of claims 287-302 to the cell.

305. A method of modifying a target sequence in a cell, the method comprising administering to the cell a composition comprising an IscB protein of any of claims 1 to 31 and / or a guide nucleic acid comprising the coRNA of any one of claims 46-174, wherein IscB protein and / or the guide nucleic acid are provided via one or more polynucleotides encoding the IscB and / or guide nucleic acid, and wherein the one or more polynucleotides are operably configured to express the IscB / or the guide nucleic acid.

306. The method of claim 304 or 305, wherein the modifying comprises cleaving a DNA polynucleotide.

307. The method of any of claims 304-306, wherein the cleaving results in 5’ overhangs.

308. A method of targeting a polynucleotide, comprising contacting a sample that comprises a target polynucleotide with the composition of any one of claims 287-302, or one or more polynucleotides encoding one or more components of the composition of any of claims 287- 302.

309. The method of claim 308, wherein contacting results in modification of a gene product or modification of the amount or expression of a gene product.

310. The method of claim 309, wherein the target sequence of the polynucleotide is a disease- associated target sequence.

311. A composition comprising: a. the IscB protein of any one of claims 1-31, wherein the IscB protein is catalytically inactive,b. a nucleotide deaminase associated with or otherwise capable of forming a complex with the IscB protein, and c. a guide nucleic acid capable of forming a complex with the IscB protein and directing site-specific binding at a target sequence.

312. A composition comprising : a. the IscB protein of any one of claims 1-31, wherein the IscB protein is catalytically inactive, b. a nucleotide deaminase associated with or otherwise capable of forming a complex with the IscB protein, and c. a guide nucleic acid comprising the coRNA of any one of claims 46-174.

313. The composition of claim 311 or 312, wherein the nucleotide deaminase is an adenosine deaminase or a cytidine deaminase.

314. A cell or progeny thereof genetically engineered to express one or more components of the composition of any one of claims 311-313.

315. A method of editing nucleic acids in target polynucleotides comprising delivering the composition of any one of claims 311-313, or one or more polynucleotides encoding one or more components of the composition, to a cell or population of cells comprising the target sequence.

316. The method of claim 315, wherein the target polynucleotides are target sequences within genomic DNA.

317. The method of claim 315 or 316, wherein the target polynucleotide is edited at one or more bases to introduce a G->A or C->T mutation.

318. An isolated cell or progeny thereof comprising one or more base edits made using the method of any one of claims 315-317.

319. A composition comprising : a. the IscB protein of any one of claims 1-31, wherein the IscB is catalytically inactive, b. a reverse transcriptase associated with or otherwise capable of forming a complex with the IscB protein, and c. a guide nucleic acid capable of forming a complex with the IscB protein and directing site-specific binding of the IscB to a target sequence, and wherein the guide nucleic further comprises a donor sequence for insertion into the target sequence.

320. A composition comprising: a. the IscB protein of any one of claims 1-31, wherein the IscB is catalytically inactive, b. a reverse transcriptase associated with or otherwise capable of forming a complex with the IscB protein, and c. a guide nucleic acid comprising the coRNA of any one of claims 46-174.

321. A method of modifying target polynucleotides comprising delivering the composition of claim 319 or 320, or one or more polynucleotides encoding one or more components of the composition to a cell, wherein the complex directs the reverse transcriptase to the target sequence and the reverse transcriptase facilitates insertion of the donor sequence from the guide nucleic acid into the target sequence322. The method of claim 321, wherein insertion of the donor sequence: introduces one or more base edits; corrects or introduces a premature stop codon; disrupts a splice site; inserts or restores a splice site; inserts a gene or gene fragment at one or both alleles of the target polynucleotide; or a combination thereof.

323. An isolated cell or progeny thereof comprising the modifications made using the method of claim 321 or 322.

324. A composition comprising: a. the IscB protein of any one of claims 1-31; b. a non-LTR retrotransposon protein associated with or otherwise capable of forming a complex with the IscB protein; c. guide nucleic acid capable of forming a complex with the IscB protein and directing site-specific binding to a target sequence; and d. a donor construct comprising a donor polynucleotide for insertion to the target sequence and located between two binding elements capable of forming a complex with the non-LTR retrotransposon protein.

325. A composition comprising: a. the IscB protein of any one of claims 1-31; b. a non-LTR retrotransposon protein associated with or otherwise capable of forming a complex with the IscB protein; c. a guide nucleic acid comprising the coRNA of any one of claims 46-174; and d. a donor construct comprising a donor polynucleotide for insertion to the target sequence and located between two binding elements capable of forming a complex with the non-LTR retrotransposon protein.

326. The composition of claim 324 or 325, wherein the IscB protein is fused to the N-terminus of the non-LTR retrotransposon protein.

327. The composition of any one of claims 324-326, wherein the IscB protein is engineered to have nickase activity.

328. The composition of claim 327, wherein the guide nucleic acid directs the fusion protein to a target sequence 5 ’ of the targeted insertion site, and wherein the IscB protein generates a double-strand break at the targeted insertion site.

329. The composition of claim 327, wherein the guide nucleic acid directs the fusion protein to a target sequence 3 ’ of the targeted insertion site, and wherein the IscB protein generates a double-strand break at the targeted insertion site.

330. The composition of any one of claims 324-329, wherein the donor polynucleotide further comprises a polymerase processing element to facilitate 3 ’ end processing of the donor polynucleotide sequence.

331. The composition of any one of claims 324-330, wherein the donor polynucleotide further comprises a homology region to the target sequence on the 5 ’ end of the donor construct, the 3’ end of the donor construct, or both.

332. The composition of any one of claims 324-331, wherein the homology region is from 8 to 25 base pairs.

333. A method of modifying target polynucleotides comprising delivering the composition of any one of claims 324-332, or one or more polynucleotides encoding one or more components of the composition to a cell, wherein the complex directs the non-LTR retrotransposon protein to the target sequence and the non-LTR retrotransposon protein facilitates insertion of the donor polynucleotide sequence from the donor construct into the target polynucleotide.

334. The method of claim 333, wherein insertion of the donor sequence: introduces one or more base edits; corrects or introduces a premature stop codon; disrupts a splice site; inserts or restores a splice site; inserts a gene or gene fragment at one or both alleles of the target polynucleotide; or a combination thereof.

335. An isolated cell or progeny thereof comprising the modifications made using the method of claim 333 or 334.

336. A method of modifying a target nucleic acid, the method comprising contacting a DNA and RNA targeting system or composition of any one of claims 185-209 or 287-302.

337. The method of claim 336, wherein the target nucleic acid is in a cell and the method comprises administering to the cell a DNA and RNA targeting system or composition of any one of claims 185-209 or 287-302.

338. The method of any one of claims 336-337, wherein said modifying the target nucleic acid comprises cleaving the target nucleic acid.

339. The method of claim 338, wherein said cleaving the target nucleic acid forms at least one 5’- overhang.

340. The method of claim 338 or 339, wherein said modifying the target nucleic acid comprises deletion, insertion or change of a nucleotide in the target nucleic acid.

341. The method of any one of claims 336-340, wherein said modifying the target nucleic acid comprises editing (e.g., changing) one or more nucleobases; correcting or introducing a premature stop codon, disrupting a splice site; inserting or restoring a splice site, inserting a gene or gene fragment into the target nucleic acid, or a combination thereof.

342. The method of any one of claims 336-341, wherein the target nucleic acid is DNA.

343. The method of claim 342, wherein the target nucleic acid is genomic DNA, episomal DNA, viral DNA, or bacterial DNA.

344. The method of claim 343, wherein the target nucleic acid is double-stranded DNA.

345. The method of claim 343, wherein the target nucleic acid is single-stranded DNA.

346. The method of any one of claims 336-341, wherein the target nucleic acid is RNA.

347. The method of claim 346, wherein the target nucleic acid is double-stranded RNA.

348. The method of claim 346, wherein the target nucleic acid is single-stranded RNA.

349. The method of claim 346, wherein the target nucleic acid is RNA selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmatic RNA (scRNA).

350. The method of any one of claims 336-349, wherein the target nucleic acid is a disease- associated nucleic acid sequence.

351. An isolated nucleic acid modified by a method of anyone of claims 336-349.

352. An isolated cell or progeny thereof comprising a nucleic acid modified by a method of anyone of claims 336-349.

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