Modified guide rnas for genome editing

Abstract

This disclosure relates to modified guide RNAs having improved in vitro and in vivo activity in genome editing methods. In some embodiments, the present disclosure provides compositions and methods for modifying a PCSK9 gene. In some aspects, the present disclosure provides a guide RNA, compositions thereof, and pharmaceutical compositions comprising a guide RNA or a composition as described herein. In some aspects, the present disclosure also provides uses and methods of using a guide RNA, a composition thereof, or a pharmaceutical composition as described herein, for inducing a double-strand break or a single-strand break in a PCSK9 gene, for reducing expression of a PCSK9 gene in a cell or subject, and for treating a patient having or at risk of having a PCSK9-related disease or condition.

Description

MODIFIED GUIDE RNAS FOR GENOME EDITINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 664,674, filed June 26, 2024, the contents of which are incorporated herein in their entirety.REFERENCE TO THE SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. The .XML copy, created on June 24, 2025, is named “01155-0078-00PCT_ST26.xml” and is 5,281,298 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.INTRODUCTION AND SUMMARY

[0003] This disclosure relates to the field of genome editing using CRISPR / Cas systems, a part of the prokaryotic immune system that recognizes and cuts exogenous genetic elements. The CRISPR / Cas system relies on a single nuclease, termed CRISPR-associated protein 9 (Cas9), which induces site-specific breaks in DNA. Cas9 is guided to specific DNA sequences by small RNA molecules termed guide RNA (gRNA). A complete guide RNA comprises tracrRNA (trRNA) and crisprRNA (crRNA). A crRNA comprising a guide region may also be referred to as a gRNA, with the understanding that to form a complete gRNA it should be or become associated covalently or noncovalently with a trRNA. The trRNA and crRNA may be contained within a single guide RNA (sgRNA) or in two separate RNA molecules of a dual guide RNA (dgRNA). Cas9 in combination with trRNA and crRNA, or with an sgRNA, is termed the Cas9 ribonucleoprotein complex (RNP).

[0004] Oligonucleotides, including sgRNAs, can be synthesized with modifications at various positions. Certain modifications and modification patterns can confer desired properties on sgRNAs, e.g., improved activity or stability. Compositions, methods, and uses including sgRNA including specific sequences and chemical modification patterns, e.g., specific scaffold sequences and chemical modification patterns, are provided here. Such sgRNA can be used to target any genomic sequence proximal to a cognate protospacer adjacent motif (PAM) for the guide.

[0005] In certain embodiments, the present disclosure provides compositions and methods for modifying a PCSK9 gene using an sgRNA including specific sequences and chemical modification patterns as described herein. In some aspects, the present disclosure provides a guide RNA, compositions thereof, and pharmaceutical compositions comprising a guide RNA or a composition as described herein. In some aspects, the present disclosure also provides uses and methods of using a guide RNA, a composition thereof, or a pharmaceutical composition as described herein, for inducing a double-strand break in a PCSK9 gene, for reducing expression of a PCSK9 gene in a cell or subject, and for treating a patient having or at risk of having a ECS' -related disease or condition. In some aspects, the present disclosure also provides uses and methods of using a guide RNA, a composition thereof, or a pharmaceutical composition as described herein, for inducing a double-strand break in a PCSK9 gene, for reducing expression of a PCSK9 gene in a cell or subject, and for treating a patient having or at risk of having a ECS' -related disease or condition.

[0006] In some embodiments, genome editing tools are provided comprising single guide RNA (sgRNA) with specific sequences and chemical modification patterns, e.g., specific scaffold sequences and chemical modification patterns. The scaffold sequences and chemical modification patterns may improve the activity of the sgRNA and the sgRNA / Spy Cas9, to cleave target DNA.

[0007] The following embodiments are encompassed.

[0008] In some embodiments, the present disclosure provides for a guide RNA (gRNA) comprising: (a) a spacer sequence of 17-20 nucleotides in length, and (b) a conserved portion comprising the nucleotide sequence of SEQ ID NO: 709 or 710, wherein (i) nucleotides 5’- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3’ or 5’- GUUUUAGACGUAGAAAUACGAAGUUAAAAU-3’ (SEQ ID NOs: 3002 and 3003, respectively) constitute a repeat / antirepeat (R / AR) region, wherein nucleotides 1-6 (LS1- LS6) and 25-30 (LS7-LS12) of the R / AR constitute a lower stem (LS) region, nucleotides 7-8 (B1-B2) and 21-24 (B3-B6) of the R / AR constitute a bulge region; and nucleotides 9-20 (US 1 -US 12) of the R / AR constitute an upper stem (US) region; (ii) nucleotides 5’- AAGGCUAGUCCGUUAUCA-3’ (SEQ ID NO: 3004) constitute a nexus region (N1-N18); (iii) nucleotides 5’-CGAAAG-3’ constitute a hairpin 1 (Hl) region (from 5’ to 3’, Hl-2, Hl-5 to Hl-8, and Hl-11); (iv) nucleotides 5’-CGAAAG-3’ constitute a hairpin 1 (Hl) region (from 5’ to 3’, Hl-2, Hl-5 to Hl-8, and Hl-11); and (v) a G nucleotide between Hl region and H2 region constitutes nucleotide n; (c) wherein the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: (i) the lower stem regioncomprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-0-Me at LS2, LS3, LS4, LS5; (ii) the bulge region comprising nucleotides not modified with 2’-0-Me at B5 and B6; (iii) the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O- Me at N6, N8, N9, N10, N13, and N14; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O- Me at Hl -5; (vi) the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; and (vii) the n is a modified or unmodified; (d) wherein the gRNA comprises a 5’ end modification.

[0009] In some embodiments, the present disclosure provides for a lipid nanoparticle (LNP) composition comprising the gRNA provided herein.

[0010] In some embodiments, the present disclosure provides for the gRNA provided herein associated with a lipid nanoparticle (LNP).

[0011] In some embodiments, the present disclosure provides for a system comprising the gRNA provided herein and an S. pyogenes (Spy) Cas9 nuclease or an mRNA which encodes a SpyCas9 nuclease.

[0012] In some embodiments, the present disclosure provides for a kit comprising the gRNA provided herein and an S. pyogenes (Spy) Cas9 nuclease or an mRNA which encodes a SpyCas9 nuclease.

[0013] In some embodiments, the present disclosure provides for a pharmaceutical formulation comprising the gRNA described herein or composition and a pharmaceutically acceptable carrier.

[0014] In some embodiments, the present disclosure provides for a method of modifying a target DNA comprising delivering an S. pyogenes (Spy) Cas9 nuclease or a nucleic acid encoding a SpyCas9 nuclease, and one or more of (i) the gRNA (ii) the composition, (iii) the system, (iv) the kit, or (v) the pharmaceutical composition described herein to a cell.

[0015] In some embodiments, the present disclosure provides for the use of the gRNA, composition, system, kit, or pharmaceutical composition described herein in the manufacture of a medicament for treating a disease or disorder.

[0016] In some embodiments, the present disclosure provides for a pharmaceutical composition comprising the guide RNA or composition described herein for inducing a double-strand break within the PCSK9 gene in a cell or reducing expression of a PCSK9 gene in a cell.

[0017] In some embodiments, the present disclosure provides for a pharmaceutical composition comprising, or use of, the guide RNA or composition described herein for treating a subject having a PCSK9 related disease.

[0018] In some embodiments, the present disclosure provides for a method of inducing a double-strand break within a PCSK9 gene in a cell or reducing expression of a PCSK9 protein in a cell comprising contacting a cell with the guide RNA and a SpyCas9 nuclease or a nucleic acid encoding a SpyCas9 nuclease wherein the nuclease has double-strand endonuclease activity; or the composition of the preceding embodiments.

[0019] In some embodiments, the present disclosure provides for a method of modifying a genomic locus in a human liver cell, the method comprising contacting a human liver cell with the guide RNA and a SpyCas9 nuclease or a nucleic acid encoding a SpyCas9 nuclease, wherein the nuclease has double-strand endonuclease activity; or the composition of the preceding embodiments.

[0020] In some embodiments, the present disclosure provides for a method of treating a PCSK9 related disease in a subject, the method comprising administering to the subject the guide RNA and a SpyCas9 nuclease or a nucleic acid encoding a SpyCas9 nuclease wherein the nuclease has double-strand endonuclease activity; or the composition or pharmaceutical composition of the preceding embodiments.

[0021] In certain embodiments, the present disclosure provides for A guide RNA (gRNA) comprising, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’, a lower stem region portion being nucleotides LSI to LS6, a bulge region portion being nucleotides Bl to B2, an upper stem region being nucleotides US1 to US12, optionally wherein the upper stem region further comprises up to 8 additional nucleotides, a bulge region portion being nucleotides B3 to B6, a lower stem region portion being nucleotides LS7 to LS12, a nexus region being nucleotides N1 to N18, and a hairpin region, wherein the hairpin region comprises, from 5’ to 3’: a hairpin 1 region being nucleotides Hl-1 to Hl-12, optionally wherein the hairpin 1 region lacks up to 6 nucleotides of Hl-1 to Hl-12, a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and a hairpin 2 region being nucleotides H2-1 to H2-15; and optionally a 3’ tail region being one or more nucleotides, wherein the gRNA comprises: a5’ end modification, modified nucleotides at 10, 11, or all nucleotides in the upper stem region, 3 or more modified nucleotides in the hairpin 2 region, wherein the gRNA further comprises one or more additional modifications selected from: modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; modified nucleotides at B2 and B3 of the bulge region; modified nucleotides at Nl, N2, N4, N7, N11, N12, and N17 of the nexus region; modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11 of the hairpin 1 region, wherein the hairpin 1 region consists of nucleotides Hl-2, Hl -5, Hl-6, Hl-7, Hl-8 and Hil l; modified nucleotides at all nucleotides of the hairpin 1 region, wherein the hairpin 1 region comprises Hl-1 to Hl-12; and modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.

[0022] In some cases, wherein the conserved portion comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 400, SEQ ID NO: 709 or SEQ ID NO: 710. In some cases, the conserved portion comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 701, or 702. In some cases, the gRNA comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 801, 802, 809, or 810. In some cases, the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, optionally further comprising nucleotides not modified with 2’-O-Me at LS6 and / or LS9; the bulge region comprising nucleotides not modified with 2’-O-Me at B5 and B6; the upper stem region comprising modified nucleotides at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, and N14, optionally further comprising a nucleotide not modified with 2’-O-Me at N16; the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl-5; the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15, optionally further comprising a modified nucleotide at H2-11; and nucleotides not modified with 2’-O- Me at H2-8, H2-9, and H2-10; and the n is a modified or unmodified nucleotide.

[0023] In some cases, the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12; and nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1. In some cases, the lower stem region comprises unmodified nucleotides at LS2, LS3, LS4, and LS5, optionally at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1. In some cases, the lower stem region comprises unmodified nucleotides at LS7, LS9, and LSI 1. In some cases, the bulge region comprises modified nucleotides at B2 and B3.In some cases, the bulge region comprises unmodified nucleotides at B5 and B6, optionally at Bl, B4, B5, and B6. In some cases, the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17. In some cases, the nexus region comprises unmodified nucleotides at N6, N8, N9, N10, N13, N14, and N18, optionally at N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18. In some cases, the hairpin 1 region comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11. In some case, the hairpin 1 region comprises an unmodified nucleotide at Hl-5. In some cases, the hairpin 2 region comprises modified nucleotides at H2- 1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-25. In some cases, the hairpin 2 region comprises unmodified nucleotides at H2-8, H2-9, and H2-10.

[0024] Embodiments herein also include guide RNA (gRNA) comprising, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’ : a lower stem region portion being nucleotides LSI to LS6, a bulge region portion being nucleotides Bl to B2, an upper stem region being nucleotides US1 to US 12, a bulge region portion being nucleotides B3 to B6, a lower stem region portion being nucleotides LS7 to LS12, a nexus region being nucleotides N1 to N18, and a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ : a hairpin 1 region being nucleotides Hl-2, Hl-5, Hl-6, Hl-7, Hl-8, and Hl-11, a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and a hairpin 2 region being nucleotides H2-1 to H2-15, and optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: a 5’ end modification, modified nucleotides at all nucleotides in the upper stem region, modified nucleotides at B2 and B3 of the bulge region; modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; modified nucleotides at Hl-2, Hl-6, Hl-7, Hl- 8, and Hl-11 of the hairpin 1 region; and modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.

[0025] In some embodiments, the lower stem region comprises unmodified nucleotides at LS2, LS3, LS4, and LS5, optionally at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1; the bulge region comprises unmodified nucleotides at B5 and B6, optionally at Bl, B4, B5, andB6; the nexus region comprises unmodified nucleotides at N6, N8, N9, N10, N13, N14, and N18, optionally atN3, N5, N6, N8, N9, N10, N13, N14, N15, and N18; the hairpin 1 region comprises an unmodified nucleotide at Hl-5; and the hairpin 2 region comprises unmodified nucleotides at H2-8, H2-9, and H2-10.

[0026] In further embodiments, the spacer sequence comprises one or more modified nucleotide; the spacer sequence comprises one or more phosphorothioate (PS) modifications; the spacer sequence comprises one or more modified nucleotide selected from a 2’-O-methyl (2’-O-Me) modified nucleotide or a 2’-fluoro (2’-F) modified nucleotide; the spacer sequence is 20 nucleotides in length; the spacer sequence comprises 2’-O-Me modified nucleotides at each of the first three 5’ terminal nucleotides (SI, S2, and S3); and / or the spacer sequence comprises one or more phosphorothioate (PS) linkages between the first four 5’ terminal nucleotides. In some cases, the spacer sequence comprises modified nucleotides at SI, S2, and S3; and nucleotides not modified with 2’-O-Me at S5, S6, S7, S12, S15, S16, and S19; optionally comprising nucleotides not modified with 2’-O-Me at S5, S6, S7, S8, S12, S15, SI 6, and SI 9. In some cases, the spacer sequence further comprises modified nucleotides at S9 and SI 3. In some cases, the spacer sequence comprises modified nucleotides at SI, S2, S3, S4, S7, S8, S9, S10, SI 1, S13, S14, S17 and S18.

[0027] In some embodiments, at least one modified nucleotide comprises a modified nucleotide that stabilizes A-form helix structure, optionally wherein each modified nucleotide comprising a 2’ -ribose modification comprises a modified nucleotide that stabilizes A-form helix structure. In some cases, each modified nucleotide comprises a modification independently selected from a 2’-O-methyl (2’-O-Me) modified nucleotide, a 2’-O-(2- methoxy ethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a bicyclic ribose analog, a locked nucleic acid (LNA), ethylene-bridged nucleic acids (ENA), a bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), and a phosphorothioate (PS) modification. In some cases, at least one modified nucleotide comprising a 2’-ribose modification further comprises a phosphorothioate modified nucleotide, optionally a 2’-O-Me modified nucleotide and a phophosphorothioate modified nucleotide. In some cases, each nucleotide not modified with 2’-O-Me is independently selected from a 2’-fluoro (2’-F) modified nucleotide and an unmodified nucleotide. In some cases, the modified nucleotides of the upper stem region are 2’-O-Me modified nucleotides. In some cases, the modified nucleotides of the lower stem region, bulge, nexus region, hairpin 1 region, or hairpin 2 region, and optionally each of the the lower stem region, bulge, nexus region, hairpin 1 region, and hairpin 2 region are 2’-O-Me modified nucleotides.

[0028] In some embodiments, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, 110, 111, 112, 122, 127, 123, 124, 128, 129, 130, 131, and 132. In some cases, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, 210, 211, 212, 122, 127, 123, 124 128, 129, 130, 131, and 132. In some cases, the conserved portion comprises a modified nucleotide sequence of SEQ ID NO: 102, 202, 122, or 222.

[0029] In some cases, the gRNA comprises a 3’ end modification. In some cases, the last two, three, or four nucleotides at the 3’ end of the gRNA are modified nucleotides. In some cases, the last three nucleotides at the 3’ end of the gRNA are modified nucleotides. In some cases, the last two, three, or four nucleotides at the 3’ end of the gRNA are 2’-O-Me modified nucleotides. In some cases, the last two, three, or four nucleotides at the 3’ end of the gRNA are linked with phosphorothioate (PS) bonds. In some cases, the last two, three, or four nucleotides at the 3’ end of the gRNA are 2’-O-Me modified nucleotides, further wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are linked with phosphorothioate (PS) bonds.

[0030] In some embodiments, the gRNA comprises a 3’ tail, optionally wherein the 3’ tail is 1, 2, 3, or 4 nucleotides, optionally 1 nucleotide in length. In some cases, the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, optionally wherein the modified nucleotide is a 2’-O-Me modified nucleotide or a phosphorothioate modified nucleotide. In some cases, the 3’ tail is fully modified. In some cases, the 3’ tail comprises a terminal 2’-O-Me modified uridine nucleotide wherein the terminal 2’-O-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage. In other cases, the gRNA does not comprise a 3’ tail.

[0031] In some cases, the first two, three, or four nucleotides at the 5’ end of the gRNA are modified nucleotides. In some cases, the first two, three, or four nucleotides at the 5’ end of the gRNA are 2’-0-Me modified nucleotides. In some cases, the first three nucleotides at the 5’ end of the gRNA are modified nucleotides. In some cases, the first two, three, or four nucleotides at the 5’ end of the gRNA are linked with phosphorothioate (PS) bonds. In some cases, the first two, three, or four nucleotides at the 5’ end of the gRNA are 2’-0-Me modified nucleotides, further wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are linked with phosphorothioate (PS) bonds.

[0032] Exemplary, but nonlimiting gRNAs herein also include, for example, a gRNA comprising a modified nucoleotide sequence selected from: (a) a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence ofSEQ ID NOs: 302, 402, 322, and 422; (b) a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 307, 407, 327, and 427; (c) a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 303, 403, 304, 404, 323, 423, 324, and 424; and (d) a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 308, 408, 309, 409, 310, 410, 311, 411, 312, 412, 328, 428, 329, 429, 330, 430, 331, 431, 332, and 432. Exemplary gRNAs also include, for example, a gRNA comprising a modified nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 302, 402, 322, and 422. In some cases, the gRNA comprises a modified nucleotide sequence selected from SEQ ID Nos: 302 and 402, and further comprising a 3’ tail, optionally wherein the 3’ tail is 1, 2, 3, or 4, nucleotides, and optionally wherein the 3’ tail comprises a terminal 2’-0-Me modified uridine nucleotide wherein the terminal 2’-0-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage. In some cases, the gRNA comprises a modified nucleotide sequence selected from SEQ ID NOs: 322 and 422. In some cases, the gRNA further comprises a spacer comprising the nucleotide sequence of SEQ ID NO: 1. Exemplary gRNAs also include those consisting of the modified nucleotide sequence of SEQ ID NOs: 322 or 422, or consisting of the modified nucleotide sequence of SEQ ID NO: 322 or 422, wherein the nucleotide sequence of the first 20 nucleotides is the sequence of SEQ ID NO: 1. Exemplary gRNAs also include a gRNA comprising the modified nucleotide sequence of any one of SEQ ID NOs: 601-619, or gRNA consisting of the modified nucleotide sequence of any one of SEQ ID NOs: 601-619. Examples include a gRNA comnprising the modified nucleotide sequence of SEQ ID NO: 602, or consisting of the modified nucleotide sequence of SEQ ID NO: 602.

[0033] Embodiments herein further include, for example, a composition comprising a gRNA described herein associated with a lipid nanoparticle (LNP), and an LNP composition comprising a gRNA described herein. An LNP may, for example, include one or more cationic lipids, helper lipids, neutral lipids, and / or stealth lipids. Embodiments herein further include compositions comprising a gRNA described herein, ora composition comprising a gRNA, such as an LNP composition, further comprising a Cas nuclease or a nucleic acid which encodes a Cas nuclease, optionally a S. pyogenes Cas9 (SpyCas9) nuclease or a nucleic acid which encodes a SpyCas9 nuclease. In some cases, the nuclease is a cleavase, nickase or dCas. In some cases, the Cas nuclease comprises an amino acid sequence at least 95%, atleast 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1089, 1092, or 1095, or the Cas nuclease comprises the amino acid sequence of SEQ ID NO: 1092. In some cases, the nucleic acid which encodes the Cas nuclease is an mRNA, for example, comprising a nucleotide sequence at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1090, 1091, 1093, or 1094, or a nucleotide sequence at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1090. In some cases, the uridine residues of the mRNA may be substituted with a modified uridine, such as one or more of Nl-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine at, for instance, at least 10%, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75- 85%, 85-95%, 90-100%, or at least 90%, or 100%. The disclosure further contemplates systems, kits, and pharmaceutical compositions comprising a gRNA or composition as described herein, as well as uses and methods for modifying a target DNA, such as comprising delivering a gRNA, composition, kit, system, or pharmaceutical composition to a cell, in vitro or in vivo, for example to a liver cell such as a hepatocyte, such as a human liver cell or human hepatocyte in vitro or in vivo.

[0034] The disclosure herein further contemplates a gRNA, composition, system, kit, or pharmaceutical composition for treating a subject having a PCSK9 related disease, or in a method of inducing a double-strand break within a PCSK9 gene in a cell or reducing expression of a PCSK9 protein in a cell, as well as a method of modifying a genomic locus in a human liver cell, and a method of treating a PCSK9 related disease in a subject, for example, by contacting a cell, such as a human liver cell, optionally in a human subject, with a gRNA, composition (e.g., LNP composition), kit, system, or pharmaceutical composition as described herein.FIGURE LEGENDS

[0035] FIG. 1 shows the results from a 12-point dose response assay to determine percent indel frequency in primary human hepatocytes at the indicated sgRNA concentrations (nM) in combination with an mRNA encoding a Spy Cas9 cleavase. The twelve guides included the same spacer sequence targeting human PCSK9 with different scaffold constant region sequences and modification patterns.

[0036] FIG. 2 shows the percent indel frequency in liver of hPCSK9 mice following in vivo delivery at the indicated total RNA amount, sgRNA and mRNA encoding Spy Cas9 cleavase.Four guides included the same spacer sequence with different scaffold constant region sequences and modification patterns.

[0037] FIG. 3 shows an exemplary sgRNA (SEQ ID NO: 399) in a possible secondary structure with labels designating individual nucleotides of the conserved region of the sgRNA, including the lower stem, bulge, upper stem, nexus (the nucleotides of which can be referred to as N1 through N18, respectively, in the 5’ to 3’ direction), and the hairpin region which includes hairpin 1 and hairpin 2 regions. A nucleotide between hairpin 1 and hairpin 2 is labeled n. A guide region may be present on an sgRNA and is indicated in this figure as “(N)x” preceding the conserved region of the sgRNA.

[0038] FIGS. 4A-4B show exemplary sgRNA modified sequences (SEQ ID NOs: 402 and 401), respectively, in a possible secondary structure. 2’-0-me modified nucleotides are circled and phosphorothioate linkages between nucleotides are shown with *..

[0039] FIG. 5 shows the percent editing (percent indels), ECso (nM), and EC90 (nM) in human embryonic kidney tissue (HEK-293) following in vitro delivery of modified sgRNAs at the indicated amount, together with mRNA encoding Spy Cas9 cleavase. Three guide RNAs targeting VEGFA included the same spacer sequence with different chemical modifications incorporated within the scaffold and spacer regions.

[0040] FIGS. 6A-6E show the percent editing (percent indels) of off-targets (OTs) (%) at 4 times the EC90 dose (4xEC9o) of three gRNAs in human embryonic kidney tissue (HEK-293) following in vitro delivery of modified sgRNAs at the indicated amount, together with mRNA encoding Spy Cas9 cleavase. The three guide RNAs (described for FIG. 5) were assessed for five low frequency off-target (OT) sites previously identified for this gRNA.

[0041] FIGS. 7A-7D show the percent editing (percent indels) of off-target (OT) (%) at 4 times the EC90 dose (4xEC9o) of three gRNAs in human embryonic kidney tissue (HEK-293) following in vitro delivery of modified sgRNAs at the indicated amount, together with mRNA encoding Spy Cas9 cleavase. The three guide RNAs (described for FIG. 5) were assessed for four high-frequency off-target (OT) sites previously identified for this gRNA.

[0042] FIG. 8 shows the coordinates of off-target (OT) sequences OT1-OT9, and the alignments between the off-target sequences (bottom) and the target sequences plus the “NRG” PAM sequence (top) — the “N” represents any nucleotide base and “R” represents a purine nucleotide base. Highlighed base pairs with an “X” symbol between the bases show base-pair mismatches in each alignment. Mismatches occuring at the 5’ end of the aligned sequences (end-clippings) are shown with a instead of an “X”.

[0043] FIG. 9 shows the results from a 12-point dose response assay to determine percent editing (frameshift frequency) and ECso in primary human hepatocytes at the indicated sgRNA concentrations (nM) in combination with an mRNA encoding a Spy Cas9 cleavase. The three guides included the same spacer sequence targeting human SERPINA1 with different scaffold constant region sequences and modification patterns.

[0044] FIG. 10 shows the percent indel frequency in liver of NTLA-Piz mice, which express mutant human SERPINA1 carrying the E342K mutation, following in vivo delivery at the indicated total RNA amount, sgRNA and mRNA encoding Spy Cas9 cleavase. Three guides were used as described for FIG. 9.DETAILED DESCRIPTION

[0045] Provided herein are modified guide RNAs (gRNAs) for use in genome editing methods.

[0046] The gRNAs provided herein are modified single guide RNAs (sgRNAs) for use in genome editing methods. Sequences of exemplary sgRNAs and sgRNA scaffolds provided herein are shown in Tables 1A-1D.

[0047] This disclosure further provides uses of these gRNAs to alter the genome of a target nucleic acid in vitro (e.g., cells cultured in vitro for use in ex vivo therapy or other uses of genetically edited cells) or in a cell in a subject such as a human (e.g., for use in in vivo therapy).Table 1A. Exemplary sgRNA scaffold sequencesTable IB. Exemplary sgRNA scaffold sequencesTable 1C. Exemplary modified sgRNA sequencesTable ID. Exemplary modified sgRNA sequences

[0048] Table 1 A shows exemplary modified sgRNA scaffold sequences with generic modified nucleotides that may stabilize A-form helix structure (indicated with lowercase) in the hairpin 2 region. Table IB shows exemplary modified sgRNA scaffold sequences. Table 1C shows exemplary modified sgRNA sequences with generic modified nucleotides that may stabilize A-form helix structure (indicated with lowercase) in the hairpin 2 region. Table ID shows exemplary modified sgRNA sequences.

[0049] The sequences of SEQ ID Nos: 101-117, 201-217, 301-317, and 401-417 shown in Tables 1A-1D lack a 3’ terminal tail. The sequences of SEQ ID Nos: 121-137, 221-237, 321- 337, and 421-437 include a 3’ modified uridine, which may be a “*mU”.

[0050]

[0051] In Tables 1C and ID and through the present disclosure, (N)i7 represents 17 consecutive nucleotides having any base wherein each N is independently a modified or unmodified nucleotide. (mN*)s represents three consecutive nucleotides each having any base, a 2’-O-Me, and a 3’ PS linkage to the next nucleotide.

[0052] As used herein through the present disclosure, including Tables 1A-1D, 4A, 4B, and 5, in the context of an unmodified sequence, A, C, G, and U are respectively an adenine, cytidine, guanine, and uridine nucleotides, wherein each nucleotide is independently unmodified or modified. In the context of a modified sequence, A, C, G, and U are respectively adenine, cytidine, guanine, and uridine nucleotides. N is a nucleotide having any base. In the absence of an indication otherwise when presented as individual nucleotides (i.e., not as (N)i7), A, C, G, U and N are RNA nucleotides, i.e., 2’-OH and 3 ’phosphodiester linkage to the 3’ adjacent nucleotide when one is present, m is indicative of a 2-O-methyl modification (2’-0-Me). f is indicative of a 2’-fluoro (2’-F) modification. As used in Tables 1 A and 1C, lowercase a, c, g, and u are indicative of modified nucleotides that may stabilize A-form helix structure, optionally a modification selected from 2’-0-me, 2’-F, 2’-O-(2’- methoxy ethyl) (2’-O-moe), locked nucleic acid (LNA), bicyclic nucleic acid (BNA), ethylene-bridged nucleic acid (ENA), and unlocked nucleic acid (UNA).

[0053] In Tables 1 A-1D, *is indicative of a phosphorothioate linkage between the nucleotides adjacent to the *. Thus, for example, mA represents 2’-O-methyl adenosine.

[0054] For SEQ ID NOs: 101-117 and 201-217, or any of the sequences shown in Tables 1 A and IB, no guide region is shown, and the length corresponds to the length of the scaffold sequence with a 20-nucleotide guide region. For SEQ ID NOs: 301-317 and 401-417, 510, or 509, or any of the seqeuences shown in Tables 1C and ID, a 20-nucleotide generic guideregion is included in the sequence, and the length corresponds to the length of the full sequence of the sgRNA. Thus, it is to be understood, for example, that the first nucleotide of the sequences shown in Tables 1A and IB, corresponds to the 21stnucleotide of the sequences shown in Tables 1C and ID.

[0055] Reference will now be made in detail to certain embodiments of the disclosure, examples of which are illustrated in the accompanying drawings. While the present teachings are described in conjunction with various embodiments, it is not intended to limit the present teachings to those embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.Definitions

[0056] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of conjugates and reference to “a cell” includes a plurality of cells (e.g., a population of cells) and the like.

[0057] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement.

[0058] The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” is not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0059] The term “or” is used in an inclusive sense in the specification, z.e., equivalent to “and / or,” unless the context clearly indicates otherwise.

[0060] The term “about”, when used before a list, modifies each member of the list. The term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0061] Ranges are understood to include the numbers at the end of the range and all logical values therebetween. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0062] At least 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing an upper limit even if one is not specifically provided as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is not specifically provided. When “at least”, “up to”, or other similar language modifies a number, it can be understood to modify each number in the series.

[0063] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0064] As used herein, ranges include both the upper and lower limit.

[0065] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.

[0066] As used herein, “detecting an analyte” and the like is understood as performing an assay in which the analyte can be detected, if present, wherein the analyte is present in an amount above the level of detection of the assay.

[0067] As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition or 100% encapsulation) that the value is limited by the method of detection. For example, 100% inhibition is understood as inhibition to a level below the level of detection of the assay, and 100% encapsulation is understood as no material intended for encapsulation can be detected outside the vesicles.

[0068] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any materialincorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls.

[0069] “Editing efficiency” or “editing percentage” or “percent editing” as used herein is the total number of sequence reads with insertions or deletions of nucleotides into the target region of interest over the total number of sequence reads following cleavage by a Cas RNP.

[0070] “Regions” as used herein describes conserved groups of nucleic acids. Regions may also be referred to as “modules” or “domains.” Regions of an sgRNA may perform particular functions, e.g., in directing endonuclease activity of the RNP, for example as described in Briner AE et al., Molecular Cell 56:333-339 (2014). Exemplary regions of an sgRNA are described in Table 3.

[0071] “Hairpin” as used herein describes a duplex of nucleic acids that is created when a nucleic acid strand folds and forms base pairs with another section of the same strand. A hairpin may form a structure that comprises a loop or a U-shape. In some embodiments, a hairpin may be comprised of an RNA loop. Hairpins can be formed with two complementary sequences in a single nucleic acid molecule bind together, with a folding or wrinkling of the molecule. In some embodiments, hairpins comprise stem or stem loop structures. As used herein, a “hairpin region” refers to hairpin 1 and hairpin 2 and the “n” between hairpin 1 and hairpin 2 of a conserved portion of an sgRNA.

[0072] “Ribonucleoprotein” (RNP) or “RNP complex” as used herein describes an sgRNA, for example, together with a nuclease, such as a Cas protein. In some embodiments, the RNP comprises Cas9 and gRNA (e.g., sgRNA, dgRNA, or crRNA).

[0073] “Stem loop” as used herein describes a secondary structure of nucleotides that form a base-paired “stem” that ends in a loop of unpaired nucleic acids. A stem may be formed when two regions of the same nucleic acid strand are at least partially complementary in sequence when read in opposite directions. “Loop” as used herein describes a region of nucleotides that do not base pair (i.e., are not complementary) that may cap a stem. A “tetraloop” describes a loop of 4 nucleotides. As used herein, the upper stem of an sgRNA may comprise a tetraloop.

[0074] “Substituted” or “Substitution” as used herein with respect to a polynucleotide refers to an alteration of a nucleobase that changes its preferred base for Watson-Crick pairing. When a certain region of a guide RNA is “unsubstituted” as used herein, the sequence of the region can be aligned to that of the corresponding conserved portion of a spyCas9 sgRNA (e.g., SEQ ID NO: 400, 709, or 710) with gaps and matches only (i.e., no mismatches), where bases are considered to match if they have the same preferred standard partner base (A, C, G, or T / U) for Watson-Crick pairing.

[0075] “Guide RNA”, “gRNA”, and “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally- occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences. Guide RNAs can include modified RNAs as described herein.

[0076] In some embodiments, the gRNA (e.g., sgRNA) comprises a “guide region”, which is sometimes referred to as a “spacer” or “spacer region,” for example, in Briner AE et al., Molecular Cell 56:333-339 (2014) for sgRNA (but applicable herein to all guide RNAs). The guide region or spacer region is also sometimes referred to as a “variable region,” “guide domain” or “targeting domain.” In some embodiments, a “guide region” immediately precedes a “conserved portion of an sgRNA” at its 5’ end, and in some embodiments the sgRNA is shortened. An exemplary “conserved portion of an sgRNA” is shown in Table 3.

[0077] Exemplary guide sequences useful in the guide RNA compositions and methods described herein are shown in Tables 1 A-1D and Table 3 and throughout the application. For example, where Tables 1 A-D and Table 3 show a guide sequence, this guide sequence may be used in a guide RNA to direct a Cas nuclease, i.e., S. pyogenes Cas9 (“spyCas9”) nuclease to a target sequence. In some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence, except for the substitution of U for T in the guide sequence.

[0078] Exemplary “conserved regions” of S. pyogenes Cas9 (“spyCas9” (also referred to as “spCas9”)) sgRNAs (SEQ ID NOs: 400, 709, and 710) are shown in Table 3. The first and second rows show the numbering of the nucleotides within each exemplary conserved region; the third to fifth rows show the sequence of SEQ ID NOs: 400, 709, and 710, respectively and the sixth row shows the regions.

[0079] A “target sequence” as used herein refers to a sequence of nucleic acid to which the guide region directs a nuclease for cleavage. In some embodiments, a spyCas9 protein may be directed by a guide region to a target sequence by the nucleotides present in the guide region. The guide region may be identical or complementary to the target sequence in the genome.

[0080] As used herein, the “5’ end” refers to the first nucleotide of the sgRNA, in which the 5’ position is not linked to another nucleotide.

[0081] As used herein, a “5’ end modification” refers to a gRNA comprising a guide region having modifications in one or more of the one (1) to about four (4) nucleotides at its 5’ end, optionally wherein the first nucleotide (from the 5’ end) of the gRNA is modified.

[0082] As used herein, the “3’ end” refers to the end or terminal nucleotide of a gRNA, in which the 3’ position is not linked to another nucleotide. In some embodiment, the 3’ end is in the 3’ tail. In some embodiments, the 3’ end is in the conserved portion of an gRNA, e.g., is part of the double stranded portion of hairpin 2. In certain embodiments, the 3’ end is in a “3’ extension” that is covalently linked to the 3’ end of the conserved portion of the gRNA.

[0083] As used herein, “3’ extension” refers to a polynucleotide containing, for example, a template sequence and a DNA-dependent DNA polymerase recruiting sequence (DRS), wherein the 3’ extension is operably linked to a spacer sequence and a scaffold sequence, e.g., a guide RNA. In certain embodiments, the 3’ extension is covalently linked to the 3’ end of the scaffold sequence. In certain embodiments, the covalent linkage is a phosphodiester bond or a phosphorothioate bond, and can include a polynucleotide linker sequence. In certain embodiments, the covalent linkage is a non-nucleotide linkage.

[0084] As used herein, a “3’ end modification” refers to a gRNA having modifications in one or more of the one (1) to about four (4) nucleotides at its 3’ end, optionally wherein the last nucleotide (i.e., the 3’ most nucleotide) of the gRNA is modified. If a 3’ tail is present, the 1- 4 nucleotides may be within, or at least partially within, the 3’ tail. If a 3’ tail is not present, the 1-4 nucleotides may be within the conserved portion of a sgRNA. If a 3’ extension is present, the nucleotides may be within the 3’ extension.

[0085] The “last,” “second to last,” “third to last,” etc., nucleotide refers to the 3’ most, second 3’ most, third 3’ most, etc., nucleotide, respectively in a given sequence. For example, in the sequence 5’-AAACTG-3’, the last, second to last, and third to last nucleotides are G, T, and C, respectively. The phrase “last 3 nucleotides” refers to the last, second to last, and third to last nucleotides; more generally, “last N nucleotides” refers to the last to the Nth to last nucleotides, inclusive. “Third nucleotide from the 3’ end of the 3’ terminus” is equivalent to “third to last nucleotide.” Similarly, “third nucleotide from the 5’ end of the 5’ terminus” is equivalent to “third nucleotide at the 5’ terminus.”

[0086] As used herein, a “protective end modification” (such as a protective 5’ end modification or protective 3’ end modification) refers to a modification of one or more nucleotides within four nucleotides of the end of an sgRNA that reduces degradation of the sgRNA, such as exonucleolytic degradation. In some embodiments, a protective end modification comprises modifications of at least two or at least three nucleotides within sevennucleotides of the end of the sgRNA. In some embodiments, the modifications comprise phosphorothioate linkages, 2’ modifications such as 2’-0-Me or 2’ -fluoro, 2’-H (DNA), ENA, UNA, or a combination thereof. In some embodiments, the modifications comprise phosphorothioate linkages and 2’-0-Me modifications. In some embodiments, three terminal nucleotides or at least three terminal nucleotides are modified, e.g., with phosphorothioate linkages or with a combination of phosphorothioate linkages and 2’-0-Me modifications. Modifications known to those of skill in the art to reduce exonucleolytic degradation are encompassed.

[0087] In some embodiments, a “3’ tail” comprises 1-20 nucleotides, typically 1-4 nucleotides, optionally 1 nucleotide, follows the conserved portion of a sgRNA at its 3’ end. As used herein, a 3’ tail performs a generic function, e.g., to provide nuclease resistance, to facilitate synthesis. A 3’ tail is not selected to provide a specific interaction with a genomic locus accessible when the spacer is bound to the target site.

[0088] “Cas nuclease”, also called “Cas protein”, as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. As used herein, “Cas9” is Spy Cas9, the variants of Spy Cas9 listed herein, known in the art, and equivalents thereof, including PAM interacting domain variants and other variants that may modulate the kinetics or binding of the nuclease to the gRNA or the gRNA-Cas9 complex to the target site. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). In certain embodiments, the Spy Cas9 further comprises a heterologous functional domain, e.g., a nuclear localization signal (NLS), a deaminase domain, a polymerase domain, a DNA binding domain.

[0089] As used herein, a first sequence is considered to “comprise a sequence with at least X% identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X% or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example,the sequence 5’-AXG where X is any modified uridine, such as pseudouridine, N1 -methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5’-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman- Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.

[0090] “mRNA” is used herein to refer to a polynucleotide that is unmodified or modified RNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof. In general, mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). An mRNA can contain modified uridines at some or all of its uridine positions.

[0091] “Messenger RNA” or “mRNA” is used herein to refer to a polynucleotide that comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise one or more chemically modified nucleosides such as 5-methyl-cytidine (5mC), 2-thio-uridine (2sU), N1 -methylpseudouridine (ml yU) and pseudo-uridine (yU), or a modified cap structure as provided below.”

[0092] As used herein, a “subject” refers to any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to nonhuman animals. In some embodiments, “subject” refers to primates. In certain embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, a subject may be a transgenic animal, genetically-engineered animal. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject”.

[0093] As used herein, “indels” refer to insertion / deletion mutations consisting of a number of nucleotides that are either inserted or deleted at the site of double-strand breaks (DSBs) in a target nucleic acid. As used herein, when indel formation results in an insertion, theinsertion is a random insertion at the site of a double-strand break and is not directed by or based on a template sequence.

[0094] As used herein, “inhibit expression” and the like refer to a decrease in expression (e.g., knockdown or knockout) of a particular gene product (e.g., protein, mRNA, or both). Expression of a protein (z.e., gene product) can be measured by detecting total cellular amount of the protein from a tissue sample, e.g., biopsy, or cell population of interest by detecting expression of a protein in individual members of a population of cells, e.g., by cell sorting to define percent of cells expressing a protein, or expression of a protein in cells in aggregate, e.g., by ELISA or western blot. Inhibition of expression can result from genetic modification of a gene sequence, e.g., a genomic sequence, such that the full-length gene product, or any gene product, is no longer detected, e.g, knockdown of the gene. Certain genetic modifications can result in the introduction of frameshift or nonsense mutations that prevent translation of the full-length gene product. Genetic modifications at a splice site, e.g, at a position sufficiently close to a splice acceptor site or a splice donor site to disrupt splicing, can prevent translation of the full-length protein. Inhibition of expression can result from a genetic modification in a regulatory sequence within the genomic sequence required for the expression of the gene product, e.g., a promoter sequence, a 3’ UTR sequence, e.g., a capping sequence, a 5’ UTR sequence, e.g., a poly A sequence. Inhibition of expression may also result from disrupting expression or activity of regulatory factors required for translation of the gene product, e.g., production of no gene product. For example, a genetic modification in a transcription factor sequence, inhibiting expression of the full-length transcription factor, can have downstream effects and inhibit expression of one or more gene products controlled by the transcription factor. Inhibition of expression can be predicted by changes in genomic or mRNA sequences. Mutations expected to result in inhibition of expression can be detected by known methods including next generation sequencing of DNA isolated from a tissue sample or cell population of interest. Inhibition of expression can be determined as the percent of cells in a population having a predetermined level of expression of a protein, z.e., a reduction of the percent or number of cells in a population expressing a protein of interest at least a certain level. Inhibition of expression can also be assessed by determining a decrease in overall protein level, e.g., in a cell or tissue sample, e.g., a biopsy sample. In certain embodiments, inhibition of expression of a secreted protein can be assessed in a fluid sample, e.g., cell culture media or a body fluid. Proteins may be present in a body fluid, e.g., blood or urine, to permit analysis of protein level. In certain embodiments, protein level may be determined by protein activity or the level of a metabolic product, e.g., in urine or blood. Insome embodiments, “inhibition of expression” may refer to some loss of expression of a particular gene product, for example a decrease in the amount of an mRNA or a protein expressed in a tissue sample or by a population of cells. In some embodiments, “inhibition” may refer to some loss of expression of a particular gene product, for example at the cell surface or secreted into a bodily fluid, e.g., blood. In some embodiments, “inhibition” may refer to some loss of expression in one, or more, cell or tissue types, but not all cell or tissue types, e.g., inhibition of expression in liver, but not in other organs. It is understood that the level of inhibition of expression is relative to a starting level, a reference level, or a control level, in the same type of subject sample. For example, routine monitoring of a protein level may be performed in a fluid sample from a subject, e.g., blood or urine, or in a tissue sample, e.g., a biopsy sample. In certain embodiments, a correlation is known, or established, wherein the level of a biomarker, e.g., in blood or urine, is correlated with the level of inhibition of expression of a target gene. It is understood that the level of inhibition of expression is for the sample being assayed. Similarly, in animal studies where serial tissue samples may be obtained, e.g, liver tissue, the target may be expressed in other tissues. Therefore, the level of inhibition of expression is not necessarily the level of inhibition of expression systemically, but within the tissue, cell type, or fluid being sampled.

[0095] As used herein, a “genetic modification” is a change at the DNA level, e.g, induced by a CRISPR / Cas9 gRNA and Cas9 system. A genetic modification may comprise an insertion, deletion, or substitution (z.e., base sequence substitution, i.e., mutation), typically within a defined sequence or genomic locus. A genetic modification changes the nucleic acid sequence of the DNA. A genetic modification may be at a single nucleotide position. A genetic modification may be at multiple nucleotides, e.g., 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, e.g., contiguous nucleotides. A genetic modification can be in a coding sequence, e.g., an exon sequence. A genetic modification can be at a splice site, i.e., sufficiently close to a splice acceptor site or a splice donor site to disrupt splicing. A genetic modification can include insertion of a nucleotide sequence not endogenous to the genomic locus, e.g., insertion of a coding sequence of a heterologous open reading frame or gene. As used herein, a genetic modification can be used to prevent translation of an endogenous full-length protein having an amino acid sequence of the full- length protein prior to genetic modification of the genomic locus. Prevention of translation of a full-length protein or gene product includes prevention of translation of a protein or gene product of any length. Translation of an endogenous full-length protein can be prevented, for example, by a frameshift mutation that results in the generation of a premature stop codon orby generation of a nonsense mutation. Translation of an endogenous full-length protein can be prevented by disruption of splicing. Translation of a full-length protein can be prevented by the insertion of a heterologous coding sequence. Translation of an endogenous full-length protein, e.g., when the endogenous full-length protein contains an unwanted mutation, can be prevented by making a change at one or more positions to change an endogenous full-length protein coding sequence to provide a modified full-length coding sequence different from the endogenous sequence present in the cell, e.g., correction of a point mutation. Translation of an endogenous full-length protein can be prevented by altering the splicing of the endogenous full-length protein to produce a different protein by alternative splicing.

[0096] “Treatment” as used herein is understood as reducing at least one sign or symptom of the disease or indication. Reduction can include to a frequency or severity such that the sign or symptom of the disease is no longer detectable. Treatment can include administration of more than one dose of the agent. Treatment can include administration with other agents. Effective treatment does not require a cure or complete elimination of the disease or indication. The rate of progression or development of a disease can be compared to the progression or development of a disease in an appropriately matched control, e.g., a population control, a control from a natural history study. As used herein, “delivering” and “administering” are used interchangeably.

[0097] Co-administration, as used herein, means that a plurality of substances are administered sufficiently close together in time so that the agents act together. Coadministration encompasses administering substances together in a single formulation and administering substances in separate formulations close enough in time so that the agents act together.

[0098] As used herein, the phrase “pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally non-toxic and is not biologically undesirable and that are not otherwise unacceptable for pharmaceutical use. Pharmaceutically acceptable generally refers to substances that are non-pyrogenic. Pharmaceutically acceptable can refer to substances that are sterile, especially for pharmaceutical substances that are for injection or infusion.

[0099] As used herein, “PCSK9” refers to the nucleic acid sequence or protein sequence of “proprotein convertase subtilisin kexin 9” or “proprotein convertase subtilisin kexin type 9.” The human wild-type PCSK9 sequence is available at NCBI Gene ID: 255738 (worldwide web at ncbi.nlm.nih.gov / gene?cmd=retrieve&dopt=default&rn=l&list_uids=255738, in the version available on the date of filing the instant application); Ensembl: ENSG00000169174MIM:607786, chrl : 55039548- chrl:55064852. Synonyms for PCSK9 include NARC1, FH3, HCHOLA3, PC9, FHCL3 and LDLCQ1. he PCSK9 gene encodes a member of the subtili sin-like proprotein convertase family, which includes proteases that process protein and peptide precursors trafficking through regulated or constitutive branches of the secretory pathway. The encoded protein undergoes an autocatalytic processing event with its prosegment in the ER and is constitutively secreted as an inactive protease into the extracellular matrix and trans-Golgi network. It is expressed in liver, intestine, and kidney tissues and escorts specific receptors for lysosomal degradation. The PCSK9 protease is involved in regulating circulating LDL cholesterol levels, and plays a role in cholesterol and fatty acid metabolism. Certain mutations or excess production of PCSK9 have been associated with cardiovascular disease and chronic liver injury. Single nucleotide polymorphisms and other variations of the human PCSK9 sequence can be found, for example, at www.ncbi.nlm. nih.gov / SNP / snp_ref.cgi?locusId=255738.

[0100] As used herein, “PCSK9 associated disorder” and the like is intended to include any disease associated with the PCSK9 gene or protein. Such a disease may be caused, for example, by excess production of the PCSK9 protein, by PCSK9 gene mutations, by abnormal cleavage of the PCSK9 protein, by abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9-associated diseases include lipidemias, e.g., a hyperlipidemias, and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases.Types of modifications described herein

[0101] Guide RNAs comprising modifications at various positions are disclosed herein. In some embodiments, a position of a gRNA that comprises a modification is modified with any one or more of the following types of modifications as denoted by the indicators in the table below. In the context of a modified sequence, in the absence of an indicator otherwise, each adenine (A), cytosine (C), guanine (G), uridine (U), or N (i.e., any nucleobase nucleotide) is understood, respectively, as an unmodified A, C, G, U, or N RNA nucleotide, i.e., 2-OH, 3 ’phosphodiester linkage to the adjacent 3’ nucleotide or a terminal 3’- phosphate.

[0102] The disclosure includes the use of nucleotides that stabilize A-form helix structures. The 2’ hydroxyl (OH) group present in RNA, but lacking in DNA, increasesmelting temperature (Tm) and stability by locking an RNA duplex into a compact A-form helix that is more stable than DNA’s standard B-form helix. The 2'-hydroxyl group prefers an axial orientation and steric and stereoelectronic effects result in the preferred C3'-endo sugar conformation or pucker. In contrast, 2 '-deoxyriboses in B-form DNA adopt a C2'-endo pucker. However, the conformational equilibrium can be shifted to the C3'-endo pucker by altering the gauche effect between 03' and 04'. Thus, the 3'-methylene and N3'^P5' phosphoramidate DNA analogs mimic RNA and prefer the C3'-endo sugar pucker. Modifications of RNA at the 2’ position of the ribose, and optionally other sugar positions, both naturally and non-naturally occurring, have long been known. Many modified nucleotides have been demonstrated to maintain and even promote A-type helix confirmation (see, e.g., Lubini et al., 1994. Stabilizing effects of the RNA 2’ -substituent: crystal structure of an oligodeoxynucleotide duplex containing 2’-0-methylated adenosines. Chemistry & Biology. 1 :39-45; Egli, 1998. Towards the structure-based design of oligonucleotide therapeutics. Adv Enzyme Regul. 38: 181-203). Modified nucleotides to promote A-form helix structures have been used in various nucleic acid-based therapeutics (see, e.g., Egli and Manoharan, 2023. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 51 : 2529-2573, doi: 10.1093 / nar / gkad067).

[0103] In the context of a modified sequence, each lowercase a, c, g, u, or n is understood as an A, C, G, U, or N modified nucleotide with a modification that stabilizes A- form helix structure, optionally a nucleotide with a modification at least at the 2’ position of the ribose, e.g.,2’-O- ethyl modified nucleotide, e.g., 2’-0(CH2)o-eCH3 modified nucleotide, e.g., 2’-O-methyl (2’-0-Me) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide; a 2’-fluoro (2’-F) modified nucleotide, bicyclic ribose analog, a locked nucleic acid (LNA), an ethylene-bridged nucleic acid (ENA), a bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), or a morpholino modified nucleotide. In certain embodiments, the a, c, g, u, or n includes a phosphorothioate modification. In certain embodiments, the phosphorothioate modification is present between at least terminal nucleotides 1 and 2 and 2 and 3 at the 3’ end of the gRNA.

[0104] In the context of an unmodified sequence, each A, C, G, U, or N is independently a modified or unmodified nucleotide.Table 2. Exemplary modifications2’-O-methyl modifications

[0105] Modified sugars are believed to control the puckering of nucleotide sugar rings, a physical property that influences oligonucleotide binding affinity for complementary strands, duplex formation, and interaction with nucleases. Substitutions on sugar rings can therefore alter the conformation and puckering of these sugars. For example, 2’-O-methyl (2’-0-Me) modifications can increase binding affinity and nuclease stability of oligonucleotides, though as shown in the Examples, the effect of any modification at a given position in an oligonucleotide needs to be empirically determined.

[0106] The terms “mA,” “mC,” “mU,” or “mG” are used to denote an A, C, U, or G nucleotide, respectively, that has been modified with 2’-0-Me.

[0107] A ribonucleotide and a modified 2’-O-methyl ribonucleotide can be depicted as follows:2’-0-(2-methoxyethyl) modifications

[0108] In some embodiments, the modification may be 2’-O-(2-methoxyethyl) (2’-O- moe). A modified 2’-O-moe ribonucleotide can be depicted as follows:

[0109] The terms “moeA,” “moeC,” “moeU,” or “moeG” are used to denote an A, C, U, or G nucleotide, respectively, that has been modified with 2’-O-moe.2’-fluoro modifications

[0110] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2’-fluoro (2’-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.

[0111] In this application, the terms “fA,” “fC,” “fU,” or “fG” are used to denote a n A, C, U, or G nucleotide, respectively, that has been modified with 2’-F.

[0112] A ribonucleotide without and with a 2’-F modification can be depicted as follows:Nature! composition of RNAPhosphorothioate modifications

[0113] A phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example between nucleotides. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

[0114] A is used to depict a PS modification. In this application, the terms A*, C*, U*, or G* are used to denote an A, C, U, or G nucleotide, respectively, that is linked tothe next (e.g., 3’) nucleotide with a PS bond. Throughout this application, PS modifications are grouped with the nucleotide whose 3’ carbon is bonded to the phosphorothioate; thus, indicating that a PS modification is at position 1 means that the phosphorothioate is bonded to the 3’ carbon of nucleotide 1 and the 5’ carbon of nucleotide 2.

[0115] In this application, the terms “mA*,” “mC*,” “mU*,” or “mG*” are used to denote an A, C, U, or G nucleotide, respectively, that has been substituted with 2’-0-Me and that is linked to the next (e.g., 3’) nucleotide with a PS linkage, which may sometimes be referred to as a “PS bond.” Similarly, the terms “fA*,” “fC*,” “fU*,” or “fG*” are used to denote a n A, C, U, or G nucleotide, respectively, that has been modified with 2’-F and that is linked to the next (e.g., 3’) nucleotide with a PS linkage. Equivalents of a PS linkage or bond are encompassed by embodiments described herein.

[0116] The diagram below shows the substitution of S- for a nonbridging phosphate oxygen, generating a PS modified linkage in lieu of a phosphodiester linkage:Natural phosphodiester Modified phosphorothioate linkage of RNA (PS) bondInverted abasic modifications

[0117] Abasic nucleotides refer to those which lack nitrogenous bases. The figure below depicts an oligonucleotide with an abasic (in this case, shown as apurinic; an abasic site could also be an apyrimidinic site, wherein the description of the abasic site is typically in reference to Watson-Crick base pairing — e.g., an apurinic site refers to a site that lacks a nitrogenous base and would typically base pair with a pyrimidinic site) site that lacks a base, wherein the base may be substituted by another moiety at the 1’ position of the furan ring (e.g., a hydroxyl group, as shown below, to form a ribose or deoxyribose site, as shown below, or a hydrogen):

[0118] Inverted bases refer to those with linkages that are inverted from the normal 5’ to 3’ linkage (i.e., either a 5’ to 5’ linkage or a 3’ to 3’ linkage). For example:Normal oligonucleotide 5 ’ -inverted oligonucleotide 3 ’ -inverted linkage with Fllinkage linkage substituted 3’inverted abasic site

[0119] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5’ nucleotide via a 5’ to 5’ linkage, or an abasic nucleotide may be attached to the terminal 3’ nucleotide via a 3’ to 3’ linkage. An inverted abasic nucleotide at either the terminal 5’ or 3’ nucleotide may also be called an inverted abasic end cap. In this application, the terms “invd” indicates an inverted abasic nucleotide linkage.Deoxyribonucleotides

[0120] A deoxyribonucleotide (in which the sugar comprises a 2’-deoxy position) is considered a modification in the context of a gRNA, in that the nucleotide is modified relative to standard RNA by the substitution of a proton for a hydroxyl at the 2’ position.Unless otherwise indicated, a deoxyribonucleotide modification at a position that is U in an unmodified RNA can also comprise replacement of the U nucleobase with a T.Bicyclic ribose analog

[0121] Exemplary bicyclic ribose analogs include locked nucleic acid (LN A), ENA, bridged nucleic acid (BNA), or another LNA-like modifications. In some instances, a bicyclic ribose analog has 2’ and 4’ positions connected through a linker. The linker can be of the formula -X-(CH2)n- where n is 1 or 2; X is O, NR, or S; and R is H or C1-3 alkyl, e.g., methyl. Examples of bicyclic ribose analogs include LNAs comprising a 2'-O-CH2-4' bicyclic structure (oxy-LNA) (see WO 98 / 39352 and WO 99 / 14226); 2'-NH-CH2-4' or 2'-N(CH3)- CH2-4' (amino-LNAs) (Singh et al., J. Org. Chem. 63: 10035-10039 (1998); Singh et al., J.Org. Chem. 63:6078-6079 (1998)); and 2'-S-CH2-4' (thio-LNA) (Singh et al., J. Org. Chem. 63:6078-6079 (1998); Kumar et al., Biorg. Med. Chem. Let. 8:2219-2222 (1998)).ENA

[0122] An ENA modification refers to a nucleotide comprising a 2'-O,4'-C-ethylene modification. An exemplary structure of an ENA nucleotide is shown below, in which wavy lines indicate connections to the adjacent nucleotides (or terminal positions as the case may be, with the understanding that if the 3’ terminal nucleotide is an ENA nucleotide, the 3’ position may comprise a hydroxyl rather than phosphate). For further discussion of ENA nucleotides, see, e.g., Koizumi et al., Nucleic Acids Res . 31 : 3267-3273 (2003).UNA

[0123] A UNA or unlocked nucleic acid modification refers to a nucleotide comprising a 2',3'-seco-RNA modification, in which the 2’ and 3’ carbons are not bonded directly to each other. An exemplary structure of a UNA nucleotide is shown below, in which wavy lines indicate connections to the adjacent phosphates or modifications replacing phosphates (or terminal positions as the case may be). For further discussion of UNAnucleotides, see, e.g., Snead et al., Molecular Therapy !'. el03, doi: 10.1038 / mtna.2013.36 (2013).Base modifications

[0124] A base modification is any modification that alters the structure of a nucleobase or its bond to the backbone, including isomerization (as in pseudouridine). In some embodiments, a base modification includes inosine. In some embodiments, a modification comprises a base modification that reduces RNA endonuclease activity, e.g., by interfering with recognition of a cleavage site by an RNase or by stabilizing an RNA structure (e.g., secondary structure) that decreases accessibility of a cleavage site to an RNase. Exemplary base modifications that can stabilize RNA structures are pseudouridine and 5-methylcytosine. See Peacock et al., J Org Chem. 76: 7295-7300 (2011). In some embodiments, a base modification can increase or decrease the melting temperature (Tm) of a nucleic acid, e.g., by increasing the hydrogen bonding in a Watson-Crick base pair, forming non-canonical base pair, or creating a mismatched base pair.Guide region

[0125] The above modifications and their equivalents are included within the scope of the embodiments described herein. In some embodiments, the gRNA further comprises a guide region. In some embodiments, the guide region comprises 17, 18, 19, or 20 nucleotides, preferably the 19 or 20 nucleotides at the 5’ end of the gRNA immediately 5’ of the conserved portion of the gRNA. In some embodiments, the guide region comprises 20 nucleotides. In some embodiments, the guide region comprises 19 nucleotides.

[0126] In some embodiments, the selection of the guide region is determined based on target sequences within the gene of interest for editing. For example, in some embodiments, the gRNA comprises a guide region that is complementary to target sequences of a gene of interest.

[0127] In some embodiments, the target sequence in the gene of interest may be complementary to the guide region of the gRNA. In some embodiments, the degree of complementarity or identity between a guide region of a gRNA and its corresponding target sequence in the gene of interest may be about 85%, 90%, 95%, or 100%. In some embodiments, the guide region of a gRNA and the target region of a gene of interest may be 100% complementary or identical. In other embodiments, the guide region of a gRNA and the target region of a gene of interest may contain at least one mismatch. For example, the guide region of a gRNA and the target sequence of a gene of interest may contain 1, 2, or 3 mismatches, where the total length of the duplex formed between the target sequence and the gRNA spacer sequence is about 20 base pairs. The 5’ terminus may comprise nucleotides that are not considered guide regions (i.e., do not function to direct a Cas9 protein to a target nucleic acid).Modifications to terminal nucleotides

[0128] In some embodiments, the 5’ or 3’ terminus regions of a gRNA are modified.3’ terminus region modifications

[0129] In some embodiments, the terminal (i.e., last) 1, 2, 3, or 4 nucleotides in the 3’ terminus region are modified. Throughout, this modification may be referred to as a “3’ end modification”. In some embodiments, the terminal (i.e., last) 1, 2, 3, or 4nucleotides in the 3’ terminus region comprise more than one modification. In some embodiments, at least three of the terminal (i.e., last) 1, 2, 3 nucleotides in the 3’ terminus region are modified. In some embodiments, the modification comprises a 2’-O-Me modification. In some embodiments, the modification comprises a PS linkage. In some embodiments, the modification comprises a 2’-O-Me modification and a PS linkage. In some embodiments, the modification to the 3’ terminus region is a 3’ protective end modification. In some embodiments, the 3’ end modification comprises a 3’ protective end modification.

[0130] In some embodiments, the 3’ end modification comprises a modified nucleotide selected from 2’-O-methyl (2’-O-Me) modified nucleotide, 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, or an inverted abasic modified nucleotide.

[0131] In some embodiments, the gRNA comprising a 3’ end modification comprises or further comprises a 3’ tail, wherein the 3’ tail comprises a modified nucleotide atany oneor more of the nucleotides present in the 3’ tail. In some embodiments, the 3’ tail is fully modified. In some embodiments, the 3’ tail is 1-4, nucleotides in length, optionally one nucleotide in length; optionally where any one or more of these nucleotides are modified. In some embodiments wherein the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, the modified nucleotide is a 2’-0-Me modified nucleotide or a phosphorothioate modified nucleotide. In some embodiments, wherein the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, the 3’ tail comprises a terminal 2’-0-Me modified uridine nucleotide wherein the terminal 2’-0-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage. In some embodiments, wherein the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, the 3’ tail is fully modified.

[0132] In some embodiments, the gRNA does not comprise a 3’ tail.

[0133] In some embodiments, the gRNA comprises a 3’ extension and does not comprise a 3’ tail.

[0134] In some embodiments, a gRNA is provided comprising a 3’ end modification, wherein the 3’ end modification comprises the 3’ end modification as shown in any one of SEQ ID NOs: 601-621. In some embodiments, a gRNA is provided comprising a 3’ protective end modification.

[0135] In some embodiments, a gRNA is provided comprising a 3’ end modification, wherein the 3’ end modification comprises (i) a 2’-O-Me modified nucleotide at the last three nucleotide of the conserved region of an gRNA and (ii) two consecutive PS linkages between the last three nucleotides of the conserved portion. In certain embodiments, the gRNA further comprises a 1 nucleotide tail, wherein the tail is a 2’-O-Me modified U linked to the 3’ end of the conserved portion with a phosphorothioate linkage.

[0136] In some embodiments, a gRNA is provided comprising a 3’ end modification, wherein the 3’ end modification comprises (i) a 2’-O-Me modified nucleotide at the last three nucleotide of the conserved region of an gRNA and (ii) three consecutive PS linkages between the last four nucleotides of the conserved portion. In certain embodiments, the gRNA further comprises a 1 nucleotide tail, wherein the tail is a 2’-O-Me modified U linked to the 3’ end of the conserved portion with a phosphorothioate linkage.

[0137] In some embodiments, the gRNA comprises a 5’ end modification and a 3’ end modification.

[0138] In some embodiments, the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are modified nucleotides. In some embodiments, the last three nucleotides at the 3’ end of the 3’ terminus are modified nucleotides. In some embodiments, the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are 2’-O-Me modified nucleotides. In some embodiments, the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are linked with phosphorothioate (PS) bonds. In some embodiments, the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are 2’-O-Me modified nucleotides, further wherein the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are linked with phosphorothioate (PS) bonds.3’ tail

[0139] In some embodiments, the gRNA comprises a 3’ terminus comprising a 3’ tail, which follows and is 3’ of the conserved portion of a gRNA. In some embodiments, the 3’ tail is about 1-10 nucleotides in length, optionally 1-4 nucleotides in length, e.g., 1 nucleotide in length.

[0140] In some embodiments, the 3’ tail comprises one or more of a protective end modification, e.g., a phosphorothioate (PS) linkage between nucleotides, a 2’-0-Me modified nucleotide, a 2’-O-moe modified nucleotide, a 2’-F modified nucleotide, or an inverted abasic modified nucleotide.

[0141] In some embodiments, the 3’ tail comprises one or more phosphorothioate (PS) linkages between nucleotides. In some embodiments, the 3’ tail comprises one or more 2’-O-Me modified nucleotides. In some embodiments, the 3’ tail comprises a combination of one or more of a phosphorothioate (PS) linkage between nucleotides and a 2’-O-Me modified nucleotide. In some embodiments, the 3’ tail is a 2’OMe modified uridine nucleotide with a phosphorothioate (PS) linkage between the tail nucleotide and the 3’ end of the conserved portion.

[0142] In some embodiments, the gRNA does not comprise a 3’ tail.

[0143] In some embodiments, the gRNA comprises a 3’ extension. In some embodiments, the gRNA comprises a 3’ extension and does not comprise a 3’ tail.

[0144] In some embodiments, the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail. In further embodiments, the modified nucleotide is a 2’-O-Me modified nucleotide or a phosphorothioate modified nucleotide.

[0145] In some embodiments, the 3’ tail comprises a terminal 2’-O-Me modified uridine wherein the terminal 2’-O-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage.5’ terminus region modifications

[0146] In some embodiments, the 5’ terminus region is modified, for example, the first 1, 2, 3, or 4 nucleotides of the gRNA are modified. Throughout, this modification may be referred to as a “5’ end modification”. In some embodiments, the first 1, 2, 3, or 4 nucleotides of the 5’ terminus region comprise more than one modification. In some embodiments, at least one of the terminal (i.e., first) 1, 2, 3, or 4 nucleotides at the 5’ end are modified. In some embodiments, at least three of the terminal 4 nucleotides at the 5’ terminus region are modified. In some embodiments, the three of the terminal nucleotides at the 5’ terminus region are modified. In some embodiments, the 5’ end modification is a 5’ protective end modification.

[0147] In some embodiments, both the 5’ and 3’ terminus regions (e.g., ends) of the gRNA are modified. In some embodiments, only the 5’ terminus region of the gRNA is modified. In some embodiments, only the 5’ terminus region of the gRNA is modified and a 3’ extension is linked to the 3’ end of the conserved portion of the gRNA.

[0148] In some embodiments, the modification(s) to the 5’ terminus are selected from a 2’-O-methyl (2’-O-Me), 2’-O-(2-methoxyethyl) (2’-O-moe) modification, or a 2’-fluoro (2’-F) modification to a nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) linkage between nucleotides. In some embodiments, the modification comprises a 2’-O-Me, 2’-O-moe, or 2’-fluoro (2’-F), modification a phosphorothioate (PS) linkage between nucleotides, e.g., 2’-O-Me modification and a phosphorothioate linkage at the 5’ terminus. In certain embodiments, the gRNA comprises three 2’-O-Me modifications at the first three nucleotides at the 5’ terminus and three phosphorothioate linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the 5’ terminus. In some embodiments, an equivalent modification is encompassed.

[0149] In some embodiments, the first two, three, or four nucleotides at the 5’ end of the gRNA are modified nucleotides. In some embodiments, the first three nucleotides at the 5’ end of the 5’ terminus are modified nucleotides. In some embodiments, the first two, three, or four nucleotides at the 5’ end of the 5’ terminus are 2’-O-Me modified nucleotides. In some embodiments, the first two, three, or four nucleotides at the 5’ end of the 5’ terminus are linked with phosphorothioate (PS) bonds. In some embodiments, the first two, three, orfour nucleotides at the 5’ end of the 5’ terminus are 2’-O-Me modified nucleotides, further wherein the first two, three, or four nucleotides at the 5’ end of the 5’ terminus are linked with phosphorothioate (PS) bonds.Exemplary modified gRNAs

[0150] In some embodiments, the conserved portion comprises a modified nucleotide. In some embodiments, the conserved portion of the gRNAs described herein comprise or consist of any of the sequences shown in Tables 1A, IB, 1C, and ID. In some embodiments, the gRNAs described herein comprise the spacer region disclosed herein and the conserved portion comprising any of the sequences shown in Tables 1A, IB, 1C, and ID.

[0151] Further, gRNAs are encompassed that comprise the modifications of any of the sequences shown in Tables 1 A-1D, and identified therein by SEQ ID No. That is, the nucleotides may be the same or different, but the modification pattern shown may be the same or similar to a modification pattern of a guide sequence of Tables 1A-1D. A modification pattern includes the relative position and identity of modifications of the gRNA (e.g. 5’ terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3’ tail region).

[0152] In some embodiments, the modification pattern contains no more than two changes, optionally no more than one change, from the modifications of any one of the sequences shown in the sequence columns of Tables 1A-1D. See also FIGS. 4A-4B (the modification pattern shown in a possible secondary structure of the guide RNA).

[0153] In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, 110,111 or 112, or of any one of SEQ ID Nos 122, 127, 123, 124, 128, 129, 130, 131 or 132. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, 210, 211 or 212, or of any one of SEQ ID Nos 222, 227, 223, 224, 228, 229, 230, 231 or 232. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 302, 307, 303, 304, 308, 309, 310,311 or 312, or of any one of SEQ ID Nos 322, 327, 323, 324, 328, 329, 330, 331 or 332. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 402, 407, 403, 404, 408, 409, 410, 411 or 412, or of any one of SEQ ID Nos 422, 427, 423, 424, 428, 429, 430, 431 or 432. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, or 110, or of any one of SEQ ID Nos: 122, 127, 123, 124, 128, 129, or 130. In someembodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, or 210, or of any one of SEQ ID Nos: 222, 227, 223, 224, 228, 229, or 230. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 302, 307, 303, 304, 308, 309, or 310, or of any one of SEQ ID Nos: 322, 327, 323, 324, 328, 329, or 330. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 402, 407, 403, 404, 408, 409, or 410, or of any one of SEQ ID Nos: 422, 427, 423, 424, 428, 429, or 430. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 102, 107, 103, and 104, or of any one of SEQ ID Nos: 122, 127, 123, or 124. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 202, 207, 203, and 204, or of any one of SEQ ID Nos: 222, 227, 223, or 224. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 302, 307, 303, and 304, or of any one of SEQ ID Nos: 322, 327, 323, or 324. In some embodiments, the modification pattern comprises the modification pattern of any one of SEQ ID NOs: 402, 407, 403, and 404, or of any one of SEQ ID Nos: 422, 427, 423, or 424.

[0154] In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 102, 202, 302, or 402; (b) SEQ ID NOs: 107, 207, 307, or 407; (c) SEQ ID NOs: 103, 203, 303, or 403; (d) SEQ ID NOs: 104, 204, 304, or 404; (e) SEQ ID NOs: 108, 208, 308, or 408; (f) SEQ ID NOs: 109, 209, 309, or 409; (g) SEQ ID NOs: 110, 210, 310, or 410; (h) SEQ ID NOs: 111, 211, 311, or 411; or (i) SEQ ID NOs: 112, 212, 312 or 412. In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 122, 222, 322, or 422; (b) SEQ ID NOs: 127, 227, 327, or 427; (c) SEQ ID NOs: 123, 223, 323, or 423; (d) SEQ ID NOs: 124, 224, 324, or 424; (e) SEQ ID NOs: 128, 228, 328, or 428; (f) SEQ ID NOs: 129, 229, 329, or 429; (g) SEQ ID NOs: 130, 230, 330, or 430; (h) SEQ ID NOs: 131, 231, 331, or 431; or (i) SEQ ID NOs: 132, 232, 332 or 432. In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 102, 202, 302, or 402; (b) SEQ ID NOs: 107, 207, 307, or 407; (c) SEQ ID NOs: 103, 203, 303, or 403; (d) SEQ ID NOs: 104, 204, 304, or 404; (e) SEQ ID NOs: 108, 208, 308, or 408; (f) SEQ ID NOs: 109, 209, 309, or 409; or (g) SEQ ID NOs: 110, 210, 310, or 410. In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 102, 202, 302, or 402; (b) SEQ ID NOs: 107, 207, 307, or 407; (c) SEQ ID NOs: 103, 203, 303, or 403; or (d) SEQ ID NOs: 104, 204, 304, or 404. In some embodiments, the modification pattern comprises themodification pattern selected from (a) SEQ ID NOs: 102, 202, 302, or 402; or (b) SEQ ID NOs: 107, 207, 307, or 407. In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 122, 222, 322, or 422; (b) SEQ ID NOs: 127, 227, 327, or 427; (c) SEQ ID NOs: 123, 223, 323, or 423; (d) SEQ ID NOs: 124, 224, 324, or 424; (e) SEQ ID NOs: 128, 228, 328, or 428; (f) SEQ ID NOs: 129, 229, 329, or 429; or (g) SEQ ID NOs: 130, 230, 330, or 430. In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 122, 222, 322, or 422; (b) SEQ ID NOs: 127, 227, 327, or 427; (c) SEQ ID NOs: 123, 223, 323, or 423; or (d) SEQ ID NOs: 124, 224, 324, or 424. In some embodiments, the modification pattern comprises the modification pattern selected from (a) SEQ ID NOs: 122, 222, 322, or 422; or (b) SEQ ID NOs: 127, 227, 327, or 427.

[0155] In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 102, 202, 302, or 402; (b) SEQ ID NOs: 107, 207, 307, or 407; (c) SEQ ID NOs: 103, 203, 303, or 403; or (d) SEQ ID NOs: 104, 204, 304, or 404. In some embodiments, the modification pattern comprises the modification pattern selected from (a) SEQ ID NOs: 102, 202, 302, or 402; or (b) SEQ ID NOs: 107, 207, 307, or 407. In some embodiments, the modification pattern comprises the modification pattern selected from: (a) SEQ ID NOs: 122, 222, 322, or 422; (b) SEQ ID NOs: 127, 227, 327, or 427; (c) SEQ ID NOs: 123, 223, 323, or 423; or (d) SEQ ID NOs: 124, 224, 324, or 424. In some embodiments, the modification pattern comprises the modification pattern selected from (a) SEQ ID NOs: 122, 222, 322, or 422; or (b) SEQ ID NOs: 127, 227, 327, or 427. sgRNAs; Domains / regions thereof

[0156] In some embodiments, a gRNA provided herein is an sgRNA. Briner AE et al. , Molecular Cell 56:333-339 (2014) describes functional domains of sgRNAs, referred to herein as “domains”, including the “spacer” domain responsible for targeting, the “lower stem”, the “bulge”, “upper stem” (which may include a tetraloop), the “nexus”, and the “hairpin 1” and “hairpin 2” domains. See Briner et al. at page 334, Figure 1 A. As described in detail elsewhere herein, one or more domains (e.g., hairpin 1 and / or the upper stem) may be shortened in an sgRNA described herein.

[0157] Table 3 and FIG. 3 provides a schematic of the domains of an sgRNA as used herein. The spacer region is not included in the table, and the length of the spacer region can vary from 17-20 nucleotides in length, with the nucleotide sequence being dependent on thetarget sequence within the genome. Modification patterns are discussed in the context of a 20-nucleotide spacer region. However, provided with the teachings herein regarding terminal modifications, modifications to the 5’ end of the gRNA with a spacer other than 20 nucleotides in length can readily be made provided with the teachings herein. For the purpose of defining modification positions herein, the spacer region is 5 ’-SI to S20 - 3’.Spacer Region

[0158] As provided herein, nucleotides SI, S2, and S3 constitute 5’ terminal nucleotides. In certain embodiments, nucleotides SI, S2, and S3 include protective modifications. In certain embodiments, nucleotides SI, S2, and S3 include 2’-0-Me and phosphorothioate modified nucleotides.

[0159] In certain embodiments, position S9 or S13 comprises a modified nucleotide. In certain embodiments, each position S9 or S13, when modified, independently comprises a 2’-0-Me modification or a 2’F modified nucleotide, optionally a 2’-0-Me modified nucleotide. In certain embodiments, S9 and S13 comprise a modified nucleotide, optionally a 2’0me modified nucleotide. In certain embodiments, S9 and S13 comprise a 2’F modified nucleotide.

[0160] In certain embodiments, position S4, S10, SI 1, SI 4, SI 7, SI 8, or S20 comprises a modified nucleotide. In certain embodiments, each position S4, S10, Si l, S14, S17, S18, or S20, when modified, independently comprises a 2’-0-Me modification or a 2’F modified nucleotide, optionally a 2’-F modified nucleotide.

[0161] In certain embodiments, positions S5, S6, S7, S8, S12, S15, S16, or S19 comprises a modified nucleotide wherein the modified nucleotide is not a 2’-0-Me modified nucleotide. In certain embodiments, each position S5, S6, S7, S8, S12, S15, S16, or S19, when modified, comprises a 2’F modified nucleotide.

[0162] In certain embodiments, positions SI, S2, S3, S4, S7, S8, S9, S10, SI 1, S13, S14, S17 and S18 comprise modified nucleotides and positions S5, S6, S12, S15, S16, S19, and S20 are unmodified.

[0163] In certain embodiments, the spacer sequence comprises modified nucleotides at SI, S2, and S3; and nucleotides not modified with 2’-0-Me at S5, S6, S7, S12, S15, S16, and SI 9. In certain embodiments, the spacer sequence comprises modified nucleotides at SI, S2, and S3; and nucleotides not modified with 2’-0-Me at S5, S6, S7, S8, S12, S15, S16, and SI 9. In certain embodiments, the spacer sequence further comprises modified nucleotides atS9 and SI 3. In certain embodiments, the spacer sequence comprises modified nucleotides at SI, S2, S3, S4, S7, S8, S9, S10, Si l, S13, S14, S17 and S18.

[0164] In some embodiments the spacer comprises a modification pattern selected from that of nucleotides 1-20 of any of the sequences shown in Table 1C or ID. In some embodiments, the spacer comprises a modification pattern selected from that of nucleotides 1-20 of (a) SEQ ID NOs: 302-304 or 402-404; (b) SEQ ID NOs: 307 and 407; and (c) SEQ ID NOs: 509, 510, 308, 408, 309, 409, 310, 410, 311, 411, 312, and 412. In some embodiments, the spacer comprises a modification pattern selected from that of nucleotides 1-20 of (a) SEQ ID NOs: 302-304 or 402-404; (b) SEQ ID NOs: 307 and 407; and (c) SEQ ID NOs: 509, 510, 308, 408, 309, 409, 310, and 410. In some embodiments, the spacer comprises a modification pattern selected from that of nucleotides 1-20 of: (a) SEQ ID NOs: 302-304 or 402-404; and (b) SEQ ID NOs: 307 and 407.

[0165] In certain embodiments, the conserved portion comprises a modified sequence of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, 110, 111, and 112. In certain embodiments, the conserved portion comprises a modified sequence of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, and 110. In certain embodiments, the conserved portion comprises a modified sequence of any one of SEQ ID NOs: 102, 107, 103, and 104. In certain embodiments, the conserved portion comprises a modified sequence of any one of SEQ ID NOs: 122, 127, 123, 124, 128, 129, 130, 131, and 132. In certain embodiments, the conserved portion comprises a modified sequence of any one of SEQ ID NOs: 122, 127, 123, 124, 128, 129, and 130. In certain embodiments, the conserved portion comprises a modified sequence of any one of SEQ ID NOs: 122, 127, 123, and 124.

[0166] In some embodiments, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, 210, 211, and 212. In some embodiments, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, and 210. In some embodiments, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, and 204. In some embodiments, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 222, 227, 223, 224, 228, 229, 230, 231, and 232. In some embodiments, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 222, 227, 223, 224, 228, 229, and 230. In some embodiments, the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 222, 227, 223, and 224.

[0167] In some embodiments, the modified nucleotide sequence of the conserved portion is selected from: (a) SEQ ID NOs: 102 and 202; (b) SEQ ID NOs: 107 and 207; (c) SEQ ID NOs: 103, 203, 104, and 204; and (d) SEQ ID NOs: 108, 208, 109, 209, 110, and 210. In some embodiments, the modified nucleotide sequence of the conserved portion is selected from: (a) SEQ ID NOs: 102 and 202; (b) SEQ ID NOs: 107 and 207; and (c) SEQ ID NOs: 103, 203, 104, and 204. In some embodiments, the modified nucleotide sequence of the conserved portion is selected from: (a) SEQ ID NOs: 122 and 222; (b) SEQ ID NOs: 127 and 227; (c) SEQ ID NOs: 123, 223, 124, and 224; and (d) SEQ ID NOs: 128, 228, 129, 229, 130, and 230. In some embodiments, the modified nucleotide sequence of the conserved portion is selected from: (a) SEQ ID NOs: 122 and 222; (b) SEQ ID NOs: 127 and 227; and (c) SEQ ID NOs: 123, 223, 124, and 224.

[0168] In some embodiments, the modified nucleotide sequence is selected from: (a) SEQ ID NOs: 302 and 402; (b) SEQ ID NOs: 307 and 407; (c) SEQ ID NOs: 303, 403, 304, and 404; and (d) SEQ ID NOs: 509, 510, 308, 408, 309, 409, 310, 410, 311, 411, 312, and 412. In some embodiments, the modified nucleotide sequence is selected from: (a) SEQ ID NOs: 302 and 402; (b) SEQ ID NOs: 307 and 407; (c) SEQ ID NOs: 303, 403, 304, and 404; and (d) SEQ ID NOs: 509, 510, 308, 408, 309, 409, 310, and 410. In some embodiments, the modified nucleotide sequence is selected from: (a) SEQ ID NOs: 302 and 402; (b) SEQ ID NOs: 307 and 407; and (c) SEQ ID NOs: 303, 403, 304, and 404. In some embodiments, the modified nucleotide sequence is selected from: (a) SEQ ID NOs: 322 and 422; (b) SEQ ID NOs: 327 and 427; (c) SEQ ID NOs: 323, 423, 324, and 424; and (d) SEQ ID NOs: 328, 428, 329, 429, 330, 430, 331, 431, 332, and 432. In some embodiments, the modified nucleotide sequence is selected from: (a) SEQ ID NOs: 322 and 422; (b) SEQ ID NOs: 327 and 427; (c) SEQ ID NOs: 323, 423, 324, and 424; and (d) SEQ ID NOs: 328, 428, 329, 429, 330, and 430. In some embodiments, the modified nucleotide sequence is selected from: (a) SEQ ID NOs: 322 and 422; (b) SEQ ID NOs: 327 and 427; and (c) SEQ ID NOs: 323, 423, 324, and 424.

[0169] In some embodiments, the gRNA comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, and 210. In some embodiments, the gRNA comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, and 204. In some embodiments, the guide RNA comprises a modified nucleotide sequence selected from (a) SEQ ID NOs: 102, 202, 302, or 402; (b) SEQ ID NOs: 107, 207, 307, or 407; (c) SEQ ID NOs: 103, 203, 303, 403; or (d) SEQ ID NOs: 104, 204, 304, or 404. In some mbodiments, the guide RNA comprises a modified nucleotide sequence selected from(a) SEQ ID NOs: 102, 202, 302, or 402; or (b) SEQ ID NOs: 107, 207, 307, or 407. In some embodiments, the gRNA comprises a modified nucleotide sequence of any one of SEQ ID NOs: 222, 227, 223, 224, 228, 229, and 230. In some embodiments, the gRNA comprises a modified nucleotide sequence of any one of SEQ ID NOs: 222, 227, 223, and 224. In some embodiments, the guide RNA comprises a modified nucleotide sequence selected from (a) SEQ ID NOs: 122, 222, 322, or 422; (b) SEQ ID NOs: 127, 227, 327, or 427; (c) SEQ ID NOs: 123, 223, 323, 423; or (d) SEQ ID NOs: 124, 224, 324, or 424. In some mbodiments, the guide RNA comprises a modified nucleotide sequence selected from (a) SEQ ID NOs: 122, 222, 322, or 422; or (b) SEQ ID NOs: 127, 227, 327, or 427.Lower stem

[0170] The sgRNA provided herein comprises a lower stem (LS) region that when viewed linearly, is separated by a bulge and upper stem regions. Exemplary sgRNAs comprising the LS region are shown in Table 3 and FIG. 3.

[0171] The lower stem region includes nucleotides LS1-LS6 (5’-GIJUUUA-3’) between the 3’ end of the spacer region and the 5’ portion of the bulge; and LS7-LS12 (5’- UAAAAU-3’), between the 3’ portion of the bulge and 5’ end of the nexus region.

[0172] In certain embodiments, embodiments, position LSI, LS8, LS10, or LS12 comprises a modified nucleotide. In certain embodiments, each position LSI, LS8, LS10, or LS12, when modified, independently comprises a 2’-O-Me modification or a 2’F modified nucleotide, optionally a 2’-O-Me modified nucleotide. In certain embodiments, LSI, LS8, LS10, and LS12 comprise modified nucleotides, optionally a 2’-O-Me modified nucleotide.

[0173] In certain embodiments, position LS6 comprises a modified nucleotide. In certain embodiments, LS6, when modified, independently comprises a 2’-O-Me modification or a 2’F modified nucleotide, optionally a 2’-F modified nucleotide.

[0174] In certain embodiments, positions LS2, LS3, LS4, LS5, LS7, LS9, or LSI 1 comprises a modified nucleotide wherein the modified nucleotide is not a 2’-O-Me modified nucleotide. In certain embodiments, each position LS2, LS3, LS4, LS5, LS7, LS9, or LSI 1, when modified, comprises a 2’F modified nucleotide. In certain embodiments, positions LS2, LS3, LS4, LS5, LS7, LS9, and LSI 1 comprise unmodified nucleotides.

[0175] In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, LS5, LS7, LS9, and LSI 1 comprise unmodified nucleotides. In certain embodiments, positions LSI, LS5, LS7, LS8, LS9, LS10,LSI 1, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, and LS6 comprise unmodified nucleotides.

[0176] In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, and LS5 comprise nucleotides not modified with 2’-0-Me. In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, and LS5 comprise unmodified nucleotides. In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, LS5, and LS6 comprise nucleotides not modified with 2’-0-Me. In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, LS5, and LS6 comprise unmodified nucleotides. In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, LS5, LS6, and LS9 comprise nucleotides not modified with 2’-0-Me. In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, LS5, LS6, and LS9 comprise unmodified nucleotides. In certain embodiments, positions LSI, LS8, LS10, and LS12 comprise modified nucleotides and positions LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1 comprise unmodified nucleotides.Bulge

[0177] The sgRNA comprises a bulge region having six nucleotides, B1-B6. Exemplary sgRNAs comprising the LS region are shown in Table 3 and FIG. 3. When viewed linearly, the bulge region is separated into two regions. The bulge region includes two nucleotides, B1-B2 (5’-GA-3’), constituting the 5’ portion of the bulge, an intervening upper stem region, and four nucleotides, B3-B6 (5’-AAGU-3’), constituting the 3’ portion of the bulge.

[0178] In certain embodiments, embodiments, position B2 or B3 comprises a modified nucleotide. In certain embodiments, each position B2 or B3, when modified, independently comprises a 2’-0-Me modification or a 2’F modified nucleotide, optionally a 2’-0-Me modified nucleotide. In certain embodiments, B2 and B3 comprise modified nucleotides, optionally a 2’-0-Me modified nucleotide.

[0179] In certain embodiments, position Bl or B4 comprises a modified nucleotide. In certain embodiments, Bl or B4, when modified, independently comprises a 2’-O-Me modification or a 2’F modified nucleotide, optionally a 2’-F modified nucleotide.

[0180] In certain embodiments, positions B5 or B6 comprises a modified nucleotide wherein the modified nucleotide is not a 2’-0-Me modified nucleotide. In certain embodiments, each position B5 or B6, when modified, comprises a 2’F modified nucleotide. In certain embodiments, positions B5 and B6 comprise unmodified nucleotides.

[0181] In certain embodiments, positions B2 and B3 comprise modified nucleotides and positions B5 and B6 comprise unmodified nucleotides. In certain embodiments, positions B2 and B3 comprise modified nucleotides and positions Bl, B4, B5, and B6 comprise unmodified nucleotides. In certain embodiments, all bulge positions comprise unmodified nucleotides.Upper stem

[0182] The sgRNA as provided in Table 3 and FIG. 3 comprises a continuous upper stem region 12 nucleotides in length, US1-US12 (5’-GCUAGAAAUAGC-3’; SEQ ID NO: 3005). The upper stem region is a stem loop sequence between the bulge portions, with the loop being a tetraloop (loop consisting of four nucleotides).

[0183] In certain embodiments at least 10 nucleotides of US 1 -US 12 are modified nucleotides. In certain embodiments at least 11 nucleotides of US1-US12 are modified nucleotides. In certain embodiments, all of the nucleotides of US1-US12 are modified nucleotides. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12 are modified with 2’-O- Me. In certain embodiments, all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12 are modified with 2’-0-Me.Nexus

[0184] The sgRNA comprises a continuous nexus region that is located 3’ of the lower stem region and 5’ of the hairpin 1 region. The nexus is 18 nucleotides in length, Nl- N18 (5’-AAGGCUAGUCCGUUAUCA-3’) as provided in Table 3 and FIG. 3.

[0185] In certain embodiments, embodiments, position Nl, N2, N4, N7, Ni l, N12, N16, or N17 comprises a modified nucleotide. In certain embodiments, each position Nl, N2, N4, N7, Ni l, N12, N16, or N17, when modified, independently comprises a 2’-0-Me modification or a 2’F modified nucleotide, optionally a 2’-0-Me modified nucleotide. In certain embodiments, Nl, N2, N4, N7, Ni l, N12, N16, and N17 comprise modified nucleotides, optionally a 2’-0-Me modified nucleotide. In certain embodiments, Nl, N2, N4,N7, Ni l, N12, and N17 comprise modified nucleotides. In certain embodiments, Nl, N2, N4, N7, N11, N12, and N17 comprise 2’ -O-Me modified nucleotides.

[0186] In certain embodiments, position N3, N5, or N15 comprises a modified nucleotide. In certain embodiments, N3, N5, or N15, when modified, independently comprises a 2’ -O-Me modification or a 2’F modified nucleotide, optionally a 2’-F modified nucleotide.

[0187] In certain embodiments, positions N6, N8, N9, N10, N13, N14, orN16 comprises a modified nucleotide wherein the modified nucleotide is not a 2’ -O-Me modified nucleotide. In certain embodiments, each position N6, N8, N9, N10, N13, N14, or N16, when modified, comprises a 2’F modified nucleotide. In certain embodiments, positions N6, N8, N9, N10, N13, N14, and N18 comprise unmodified nucleotides. In certain embodiments, N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18 comprise unmodified nucleotides.

[0188] In certain embodiments, positions Nl, N2, N4, N7, Ni l, N12, N16, and N17 comprise modified nucleotides and positions N6, N8, N9, N10, N13, N14, and N18 comprise unmodified nucleotides. In certain embodiments, positions Nl, N2, N4, N7, Nl 1, N12, N16, and N17 positions comprise modified nucleotides and positions N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18 comprise unmodified nucleotides. In certain embodiments, positions Nl, N2, N4, N7, Ni l, N12, N14, and N17 positions comprise modified nucleotides, and positions N3, N5, N6, N8, N9, N10, N13, N15, N16, and N18 positions comprise unmodified nucleotides. In certain embodiments, positions Nl, N2, N4, N7, Nl 1, N12, and N17 comprise modified nucleotides, and positions N3, N5, N6, N8, N9, N10, N13, N14, N15, N16, and N18 comprise unmodified nucleotides. In certain embodiments, the nexus region is fully unmodified. In certain embodiments, positions Nl, N2, N4, N7, Nl 1, N12, and N17 comprise modified nucleotides, and positions N6, N8, N9, N10, N13, and N14 are not modified with 2’-O-Me. In certain embodiments, positions Nl, N2, N4, N7, Nl 1, N12, and N17 comprise modified nucleotides, and positions N6, N8, N9, N10, N13, and N14 are not modified. In certain embodiments, positions Nl, N2, N4, N7, Ni l, N12, and N17 comprise modified nucleotides, and positions N6, N8, N9, N10, N13, N14, and N16 are not modified with 2’-O-Me. In certain embodiments, positions Nl, N2, N4, N7, Ni l, N12, and N17 comprise modified nucleotides, and positions N6, N8, N9, N10, N13, N14, and N16 are not modified. In certain embodiments, positions Nl, N2, N4, N7, N11, N12, N16, and N17 comprise modified nucleotides, and positions N6, N8, N9, N10, N13, N14, and N16 are not modified with 2’ -O-Me.Hairpin

[0189] The sgRNA disclosed herein comprises two hairpin regions 5’ of the nexus region, hairpin 1 (Hl) and hairpin 2 (H2), with a single nucleotide designated as “n” between the hairpins. Hairpin 1 as provided in Table 3 (see also FIG. 3), SEQ ID NO: 400, is 12 nucleotides in length, Hl-1 to Hl-12 (5’-ACUUGAAAAAGU-3’; SEQ ID NO: 3006) with a stem loop with a four base pair duplex and a tetraloop. In preferred embodiments provided herein, Hairpin 1 is shortened and includes positions Hl-1, Hl -2, Hl -5 - Hl -8, Hl-11, and Hl-12 (5’-ACGAAAGU-3’) as shown in Table 3. In other preferred embodiments provided herein, Hairpin 1 is shortened and includes positions Hl -2, Hl -5 - Hl -8, and Hl-11 (5’- CGAAAG). Thus, in some embodiments, Hairpin 1 lacks 4-6 nucleotides of those shown in Table 3 and FIG. 3. In some embodiments, Hairpin 1 lacks 6 nucleotides of those shown in Table 3 and FIG. 3 and SEQ ID NO: 400, and is therefore 6 nucleotides in length. See, e.g., SEQ ID Nos: 709 and 710 shown in Table 3. Hairpin 2 as provided in Table 3 and Figure 3 is 15 nucleotides in length, H2-1 to H2-15 (5’-GCACCGAGUCGGUGC-3’; SEQ ID NO: 3007). The intervening nucleotide n is a G. H2-15 is the 3’ terminal nucleotide of the constant region of the gRNA. HP2 is a stem loop with a 6 base pair stem and a 3-nucleotide loop.

[0190] In certain embodiments, position Hl-1, Hl-6, Hl-7, Hl-8, or Hl-11 comprises a modified nucleotide. In certain embodiments, each position Hl-1, Hl-6, Hl-7, Hl-8, or Hil l, when modified, independently comprises a 2’-0-Me modification or a 2’F modified nucleotide, optionally a 2’-0-Me modified nucleotide. In certain embodiments, Hl-1, Hl-6, Hl-7, Hl-8, and Hl-11 comprise modified nucleotides, optionally 2’-0-Me modified nucleotides.

[0191] In certain embodiments, position Hl -5 comprises a modified nucleotide wherein the modified nucleotide is not a 2’-0-Me modified nucleotide. In certain embodiments, Hl-5, when modified, comprises a 2’F modified nucleotide. In certain embodiments, position Hl-5 comprise an unmodified nucleotide.

[0192] In certain embodiments, positions Hl-1, Hl-6, Hl-7, Hl-8, and Hl-11 comprise modified nucleotides and position Hl-5 comprises an unmodified nucleotide. In certain embodiments, hairpin 1 comprises no modified nucleotides. In certain embodiments, positions Hl -2, Hl-6, Hl-7, Hl-8, and Hl-11 comprise modified nucletodies and position Hl-5 comprises a nucleotide that is not modified with 2’-0-Me. In certain embodiments, positions Hl -2, Hl-6, Hl-7, Hl-8, and Hl-11 comprise modified nucletodies and position Hl-5 comprises a nucleotide that is not modified.

[0193] In certain embodiments, embodiments, position H2-1, H2-2, H2-3, H2-4, H2- 5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, or H2-15 comprises a modified nucleotide. In certain embodiments, each position H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2- 12, H2-13, H2-14, or H2-15, when modified, independently comprises a 2’-O-Me modification or a 2’F modified nucleotide, optionally a 2’-O-Me modified nucleotide. In certain embodiments, H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2- 14, and H2-15 comprise modified nucleotides, optionally a 2’-O-Me modified nucleotide.

[0194] In certain embodiments, position H2-8, H2-9, or H2-10 comprises a modified nucleotide. In certain embodiments, H2-8, H2-9, or H2-10, when modified, independently comprises a 2’-O-Me modification or a 2’F modified nucleotide, optionally a 2’-F modified nucleotide. In certain embodiments, H2-8, H2-9, and H2-10 comprise modified nucleotides. In certain embodimments, H2-8, H2-9, and H2-10 comprise unmodified nucleotides.

[0195] In certain embodiments, positions H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15 comprise modified nucleotides and positions H2-8, H2-9, and H2-10 comprise unmodified nucleotides. In certain embodiments, positions H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 comprise modified nucleotides and positions H2-8, H2-9, and H2-10 comprise a nucleotide that is not modified with 2’-O-Me. In certain embodiments, positions H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 comprise modified nucleotides and positions H2-8, H2-9, and H2-10 comprise a nucleotide that is not modified. In certain embodiments, positions H2- 1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15 comprise modified nucleotides and positions H2-8, H2-9, and H2-10 comprise a nucleotide that is not modified with 2’-O-Me. In certain embodiments, positions H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15 comprise modified nucleotides and positions H2-8, H2-9, and H2-10 comprise a nucleotide that is not modified.

[0196] In certain embodiments, position n is a modified nucleotide. In certain embodiments, n is a 2’-0-Me modified nucleotide. In certain embodiments, n is an unmodified nucleotide.3’ terminus

[0197] The sgRNA has a 3’ end, which is the last nucleotide of the sgRNA. The 3’ terminus region includes the last 1-4 nucleotides from the 3’ end. In some embodiments, the 3’ end is the end of hairpin 2. In some embodiments, the sgRNA comprises nucleotides after the hairpin region(s). In some embodiments, the sgRNA includes a 3’ tail region, in whichcase the last nucleotide of the 3’ tail is the 3’ terminus. In some embodiments, the 3’ tail region comprises 1, 2, 3, or 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3’ extension is present. 3’ terminus modifications are provided above.

[0198] In some embodiments, the present disclosure provides for a guide RNA (gRNA) comprising: (a) a spacer sequence of 17-20 nucleotides in length, and (b) a conserved portion comprising the nucleotide sequence of SEQ ID NO: 709 or 710, wherein (i) nucleotides 5’-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3’ or 5’- GUUUUAGACGUAGAAAUACGAAGUUAAAAU-3’ constitute a repeat / antirepeat (R / AR) region, wherein nucleotides 1-6 (LS1-LS6) and 25-30 (LS7-LS12) of the R / AR constitute a lower stem (LS) region, nucleotides 7-8 (B1-B2) and 21-24 (B3-B6) of the R / AR constitute a bulge region; and nucleotides 9-20 (US 1 -US 12) of the R / AR constitute an upper stem (US) region; (ii) nucleotides 5’-AAGGCUAGUCCGUUAUCA-3’ constitute a nexus region (N1-N18); (iii) nucleotides 5’-CGAAAG-3’ constitute a hairpin 1 (Hl) region (from 5’ to 3’, Hl-2, Hl-5 to Hl-8, and Hl-11); (iv) nucleotides 5’-CGAAAG-3’ constitute a hairpin 1 (Hl) region (from 5’ to 3’, Hl-2, Hl-5 to Hl-8, and Hl-11); and (v) a G nucleotide between Hl region and H2 region constitutes nucleotide n; (c) wherein the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: (i) the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-0-Me at LS2, LS3, LS4, LS5; (ii) the bulge region comprising nucleotides not modified with 2’-0-Me at B5 and B6; (iii) the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US 10, US 11, and US 12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’ -O-Me at N6, N8, N9, N10, N13, and N14; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-0-Me at Hl-5; (vi) the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’ -O-Me at H2-8, H2-9, and H2-10; and (vii) the n is a modified or unmodified; (d) wherein the gRNA comprises a 5’ end modification.

[0199] In some embodiments, the present disclosure provides for a guide RNA (gRNA) comprising: (a) a spacer sequence of 17-20 nucleotides in length, and (b) a conserved portion comprising the nucleotide sequence of SEQ ID NO: 709 or 710, wherein(i) nucleotides 5’-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3’ or 5’- GUUUUAGACGUAGAAAUACGAAGUUAAAAU-3’ constitute a repeat / antirepeat (R / AR) region, wherein nucleotides 1-6 (LS1-LS6) and 25-30 (LS7-LS12) of the R / AR constitute a lower stem (LS) region, nucleotides 7-8 (B1-B2) and 21-24 (B3-B6) of the R / AR constitute a bulge region; and nucleotides 9-20 (US 1 -US 12) of the R / AR constitute an upper stem (US) region; (ii) nucleotides 5’-AAGGCUAGUCCGUUAUCA-3’ constitute a nexus region (N1-N18); (iii) nucleotides 5’-CGAAAG-3’ constitute a hairpin 1 (Hl) region (from 5’ to 3’, Hl-2, Hl-5 to Hl-8, and Hl-11); (iv) nucleotides 5’-CGAAAG-3’ constitute a hairpin 1 (Hl) region (from 5’ to 3’, Hl-2, Hl-5 to Hl-8, and Hl-11); and (v) a G nucleotide between Hl region and H2 region constitutes nucleotide n.

[0200] In some embodiments, the spacer sequence comprises 17, 18, 19, or 20 nucleotides in length. In some embodiments, the spacer sequence comprises 17 nucleotides in length. In some embodiments, the spacer sequence comprises 18 nucleotides in length. In some embodiments, the spacer sequence comprises 19 nucleotides in length. In some embodiments, the spacer sequence comprises 20 nucleotides in length.

[0201] In some embodiments, the conserved portion comprises the nucleotide sequence of SEQ ID NO: 709. In some embodiments, the conserved portion comprises the nucleotide sequence of SEQ ID NO: 710.

[0202] In some embodiments, the conserved portion comprises at least one, two, three, four, five, six, or seven regions with unmodified and modified nucleotides selected from: (i) the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5; (ii) the bulge region comprising nucleotides not modified with 2’-O-Me at B5 and B6; (iii) the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US 10, US 11, and US 12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’ -O-Me at N6, N8, N9, N10, N13, and N14; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl-5; (vi) the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’ -O-Me at H2-8, H2-9, and H2-10; and (vii) the n is a modified or unmodified; (d) wherein the gRNA comprises a 5’ end modification. In further embodiments, the conserved portion comprises at least one region with unmodified andmodified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least two regions with unmodified and modified nucleotides selected from (i)- (vii). In some embodiments, the conserved portion comprises at least three regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least four regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least five regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least six regions with unmodified and modified nucleotides selected from (i)-(vii).

[0203] In some embodiments, the conserved portion comprises one region with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises two regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises three regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises four regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises five regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises six regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises seven regions with unmodified and modified nucleotides selected from (i)-(vii).

[0204] In some embodiments, the conserved portion does not comprise the hairpin 2 (H2) region with unmodified and modified nucleotides according to (v).

[0205] In some embodiments, the conserved portion comprises one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises two regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises five regions with unmodified and modified nucleotides selected from (i)-(v).

[0206] In some embodiments, wherein the conserved portion comprises the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; and the conserved portion further comprises at least one, two, three, four, or fiveregions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least two regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least five regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises two regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises five regions with unmodified and modified nucleotides selected from (i)-(v).

[0207] In some embodiments, the conserved portion comprises (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’ -O-Me at N6, N8, N9, N10, N13, and N14; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl-5. In some embodiments, the conserved portion comprises (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, and N14; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hil l; and nucleotides not modified with 2’ -O-Me at Hl-5, and further comprises at least one, two, or three regions with unmodified and modified nucleotides selected from (i)-(iii) and(vi).

[0208] In some embodiments, the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12. In some embodiments, the lower stem region comprises nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5. In some embodiments, the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12; and nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5.

[0209] In some embodiments, the bulge region comprises nucleotides not modified with 2’-O-Me at B5 and B6. In some embodiments, the upper stem region comprises modified nucleotides at 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the upper stem region comprises modified nucleotides at 11 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the upper stem region comprises modified nucleotides at 12 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the upper stem region comprises modified nucleotides at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17. In some embodiments, the nexus region comprises nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, and N14. In some embodiments, the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, and N14. In some embodiments, the hairpin 1 (Hl) region comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11. In some embodiments, the hairpin 1 (Hl) region comprises nucleotides not modified with 2’-O-Me at Hl -5. In some embodiments, the hairpin 1 (Hl) region comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl -5. In some embodiments, the hairpin 2 (H2) region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15. In some embodiments, the hairpin 2 (H2) region comprises nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10. In some embodiments, the hairpin 2 (H2) region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2- 6, H2-7, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10. In some embodiments, the n is a modified nucleotide. In some embodiments, the n is an unmodified nucleotide.

[0210] In some embodiments, the conserved portion comprises at least one, two, three, four, five, six, or seven regions with unmodified and modified nucleotides selected from: (i) the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, and LS6; (ii) the bulge region comprising nucleotides not modified with 2’-O-Me at B5 and B6; (iii) the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; (iv) the nexus regioncomprising modified nucleotides atNl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-0-Me at N6, N8, N9, N10, N13, N14, and N16; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-0-Me at Hl-5; (vi) the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-0-Me at H2-8, H2-9, and H2-10; and (vii) the n is a modified or unmodified nucleotide. In some embodiments, the conserved portion comprises at least one region with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least two regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least three regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least four regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least five regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least six regions with unmodified and modified nucleotides selected from (i)- (vii).

[0211] In some embodiments, the conserved portion comprises one region with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises two regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises three regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises four regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises five regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises six regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises seven regions with unmodified and modified nucleotides selected from (i)-(vii).

[0212] In some embodiments, the conserved portion does not comprise the hairpin 2 (H2) region with unmodified and modified nucleotides according to (vi).

[0213] In some embodiments, the conserved portion comprises one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises two regions with unmodified and modified nucleotides selected from (i)-(vi). In some embodiments, the conserved portion comprises three regions withunmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises five regions with unmodified and modified nucleotides selected from (i)-(v).

[0214] In some embodiments, wherein the conserved portion comprises the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2- 8, H2-9, and H2-10; and the conserved portion further comprises at least one, two, three, four, or five regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least two regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least five regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises two regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises five regions with unmodified and modified nucleotides selected from (i)- (v).

[0215] In some embodiments, the conserved portion comprises (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16; and (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl -5. In some embodiments, the conserved portion comprises (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16; and (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6,Hl -7, Hl -8, and Hl-11; and nucleotides not modified with 2’-0-Me at Hl -5, and further comprises at least one, two, or three regions with unmodified and modified nucleotides selected from (i)-(iii) and (vi).

[0216] In some embodiments, the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12. In some embodiments, the lower stem region comprises nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, and LS6. In some embodiments, the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, and LS6. In some embodiments, the bulge region comprises nucleotides not modified with 2’-O-Me at B5 and B6. In some embodiments, the upper stem region comprises modified nucleotides at 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US 12. In some embodiments, the upper stem region comprises modified nucleotides at 11 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US 12. In some embodiments, the upper stem region comprises modified nucleotides at 12 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US 12. In some embodiments, the upper stem region comprises modified nucleotides at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17. In some embodiments, the nexus region comprises nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16. In some embodiments, the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16. In some embodiments, the hairpin 1 (Hl) region comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11. In some embodiments, the hairpin 1 (Hl) region comprises nucleotides not modified with 2’-O-Me at Hl-5. In some embodiments, the hairpin 1 (Hl) region comprises modified nucleotides at Hl -2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl-5. In some embodiments, the hairpin 2 (H2) region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15. In some embodiments, the hairpin 2 (H2) region comprises nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10. In some embodiments, the hairpin 2 (H2) region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2- 12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10. In some embodiments, the n is a modified nucleotide. In some embodiments, the n is an unmodified nucleotide.

[0217] In some embodiments, the conserved portion comprises at least one, two, three, four, five, six, or seven regions with unmodified and modified nucleotides selected from: (i) the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, LS6, and LS9; (ii) the bulge region comprising nucleotides not modified with 2’-O-Me at B5 and B6; (iii) the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, N16, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hil l; and nucleotides not modified with 2’-O-Me at Hl-5; (vi) the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2- 12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; and (vii) the n is a modified or unmodified nucleotide. In some embodiments, the conserved portion comprises at least one region with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least two regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least three regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least four regions with unmodified and modified nucleotides selected from (i)- (vii). In some embodiments, the conserved portion comprises at least five regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises at least six regions with unmodified and modified nucleotides selected from (i)-(vii).

[0218] In some embodiments, the conserved portion comprises one region with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises two regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises three regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises four regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises five regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises six regions with unmodified and modified nucleotides selectedfrom (i)-(vii). In some embodiments, the conserved portion comprises seven regions with unmodified and modified nucleotides selected from (i)-(vii).

[0219] In some embodiments, the conserved portion does not comprise the hairpin 2 (H2) region with unmodified and modified nucleotides according to (v).

[0220] In some embodiments, the conserved portion comprises one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises two regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion comprises four regions with unmodified and modified nucleotides selected from (i)-(vii). In some embodiments, the conserved portion comprises five regions with unmodified and modified nucleotides selected from (i)-(v).

[0221] In some embodiments, wherein the conserved portion comprises the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2- 8, H2-9, and H2-10; and the conserved portion further comprises at least one, two, three, four, or five regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least two regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises at least five regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises one region with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises two regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises three regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises four regions with unmodified and modified nucleotides selected from (i)-(v). In some embodiments, the conserved portion further comprises five regions with unmodified and modified nucleotides selected from (i)- (v).

[0222] In some embodiments, the conserved portion comprises (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16; and (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl -5. In some embodiments, the conserved portion comprises (iv) the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16; (v) the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl- 7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl-5, and further comprises at least one, two, or three regions with unmodified and modified nucleotides selected from (i)-(iii).

[0223] In some embodiments, the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12. In some embodiments, the lower stem comprises nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, LS6, and LS9. In some embodiments, the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, LS6, and LS9. In some embodiments, the bulge region comprises nucleotides not modified with 2’-O-Me at B5 and B6. In some embodiments, the upper stem region comprises modified nucleotides at 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the upper stem region comprises modified nucleotides at 11 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the upper stem region comprises modified nucleotides at 12 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the upper stem region comprises modified nucleotides at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12. In some embodiments, the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, N16, and N17. In some embodiments, the nexus region comprises nucleotides not modified with 2’-O- Me at N6, N8, N9, N10, N13, N14, and N16. In some embodiments, the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, N16, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16. In some embodiments, the hairpin 1 (Hl) region comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11. In some embodiments, the hairpin 1 (Hl) region comprises nucleotides not modified with 2’-O-Me at Hl-5. In some embodiments, the hairpin 1 (Hl) region comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modifiedwith 2’-0-Me at Hl -5. In some embodiments, the hairpin 2 (H2) region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15. In some embodiments, the hairpin 2 (H2) region comprises nucleotides not modified with 2’-0-Me at H2-8, H2-9, and H2-10. In some embodiments, the hairpin 2 (H2) region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2- 12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-0-Me at H2-8, H2-9, and H2-10. In some embodiments, the n comprises a modified nucleotide. In some embodiments, the n comprises an unmodified nucleotide.Table 3. Exemplary Conserved Portion of a spyCas9 sgRNA (SEQ ID NOs:400, 709, 710 are provided in the 3rd’ 4th, 5throws respectively)

[0224] In some embodiments, a gRNA herein comprises, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’: a. a lower stem region portion being nucleotides LSI to LS6, b. a bulge region portion being nucleotides B 1 to B2, c. an upper stem region being nucleotides US1 to US 12, optionally wherein the upper stem region further comprises up to 8 additional nucleotides, d. a bulge region portion being nucleotides B3 to B6, e. a lower stem region portion being nucleotides LS7 to LS12, f. a nexus region being nucleotides N1 to N18, and g. a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ : i. a hairpin 1 region being nucleotides Hl-1 to Hl-12, optionally wherein the hairpin 1 region lacks up to 6 nucleotides of Hl-1 to Hl-12, ii. a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and iii. a hairpin 2 region being nucleotides H2-1 to H2-15; and h. optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: i. a 5’ end modification, j. modified nucleotides at 10, 11, or all nucleotides in the upper stem region, k. 3 or more modified nucleotides in the hairpin 2 region, wherein the gRNA further comprises one or more additional modifications selected from: l. modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; m. modified nucleotides at B2 and B3 of the bulge region; n. modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; o. modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11 of the hairpin 1 region, wherein the hairpin 1 region consists of nucleotides Hl-2, Hl -5, Hl-6, Hl-7, Hl-8 and Hl-11; p. modified nucleotides at all nucleotides of the hairpin 1 region, wherein the hairpin 1 region comprises Hl-1 to Hl-12; andq. modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.In some such embodiments, the conserved portion has a nucleotide sequence as shown in Table 3 above of SEQ ID NO: 400, wherein the hairpin 1 region consists of all of Hl-1 to Hl-12. In other embodiments, the conserved portion comprises a nucleotide sequence of SEQ ID NO: 709 or 710, wherein the hairpin 1 region consists of Hl-2, Hl-5, Hl-6, Hl-7, Hl -8 and Hl-11. In some embodiments, the conserved portion comprises a nucleotide sequence of SEQ ID NO: 701 or 702. In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 102, 103, 104, 105, 106, 107, 115, or 116, or 202, 203, 204, 205, 206, 207, 215, or 216, optionally further comprising a 3’ tail as described herein. In some embodiments, the gRNA comprises the modified nucleotide sequence of SEQ ID NO: 302, 303, 304, 305, 306, or 307 or 402, 403, 404, 405, 406, or 407, optionally further comprising a 3’ tail as described herein. In some embodiments, the gRNA conserved portion includes a 3’ modified urididine and consists of the modified nucleotide sequence of SEQ ID NO: 122, 123, 124, 125, 126, 127, 135, or 136, or 222, 223, 224, 225, 226, 227, 235, or 236. In some embodiments, the gRNA consists of the modified nucleotide sequence of SEQ ID NO: 322, 323, 324, 325, 326, or 327 or 422, 423, 424, 425, 426, or 427, which includes a 3’ modified uridine. In other cases, the hairpin 1 region comprises all of Hl-1 to Hl-12 wherein all of Hl-1 to Hl-12 are modified. In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 114 or 214, and optionally further comprising a 3’ tail as described herein. In some embodiments, the gRNA conserved portion comprises or consists of the modified nucleotide sequence of SEQ ID NO: 134 or 234. In some cases, the upper stem region comprises 8 additional nucleotides and 4 additional base pairs compared to SEQ ID NO: 400. In some such cases, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 115, 116, 215, or 216, optionally further comprising a 3’ tail as described herein. In some such cases, the gRNA conserved portion comprises or consists of the modified nucleotide sequence of SEQ ID NO: 135, 136, 235, or 236.

[0225] In some embodiments, the guide RNA (gRNA) comprises, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’ : a. a lower stem region portion being nucleotides LSI to LS6, b. a bulge region portion being nucleotides B 1 to B2,c. an upper stem region being nucleotides US 1 to US 12, d. a bulge region portion being nucleotides B3 to B6, e. a lower stem region portion being nucleotides LS7 to LS12, f. a nexus region being nucleotides N1 to N18, and g. a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ : i. a hairpin 1 region being nucleotides Hl-1 to Hl-12, or alternatively a hairpin 1 region being nucleotides Hl-2, Hl-5, Hl-6, Hl-7, Hl-8, and Hl-11, ii. a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and iii. a hairpin 2 region being nucleotides H2-1 to H2-15, and h. optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: i. a 5’ end modification, j . modified nucleotides at all nucleotides in the upper stem region, k. modified nucleotides at B2 and B3 of the bulge region; l. modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; m. modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; and n. modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.In some cases, the hairpin 1 region consists of all of Hl-1 to Hl-12. Thus, in some such embodiments, the conserved portion has a nucleotide sequence as shown in Table 3 above of SEQ ID NO: 400, wherein the hairpin 1 region consists of all of Hl-1 to Hl-12. In some cases, all of Hl-1 to Hl-12 are modified. In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 114 or 214, and optionally further comprising a 3’ tail as described herein. In other cases, the hairpin 1 region consists of nucleotides Hl-2, Hl-5 to Hl-8 and Hl-11 and comprises no modified nucleotides. In other cases, the hairpin 1 region consists of Hl-2, Hl-5 to Hl-8 and Hl-11 and comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11. In some such embodiments, the conserved portion comprises a nucleotide sequence of SEQ ID NO: 709 or 710, wherein the hairpin 1 region consists of Hl -2, Hl-5, Hl-6, Hl-7, Hl-8 and Hl-11. In some embodiments, the conserved portion comprises a nucleotide sequence of SEQ ID NO:701 or 702. In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 102, 103, 104, 105, 106, or 107, or 202, 203, 204, 205, 206, or 207, optionally further comprising a 3’ tail as described herein. In some embodiments, the gRNA comprises the modified nucleotide sequence of SEQ ID NO: 302, 303, 304, 305, 306, or 307 or 402, 403, 404, 405, 406, or 407, optionally further comprising a 3’ tail as described herein. In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 122, 123, 124, 125, 126, or 127, or 222, 223, 224, 225, 226, or 227. In some embodiments, the gRNA conserved portion consists of the modified nucleotide sequence of SEQ ID NO: 122, 123, 124, 125, 126, or 127, or 222, 223, 224, 225, 226, or 227. In some embodiments, the gRNA comprises the modified nucleotide sequence of SEQ ID NO: 322, 323, 324, 325, 326, or 327 or 422, 423, 424, 425, 426, or 427. In some embodiments, the gRNA consists of the modified nucleotide sequence of SEQ ID NO: 322, 323, 324, 325, 326, or 327 or 422, 423, 424, 425, 426, or 427.

[0226] In some embodiments, the guide RNA (gRNA) comprises, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’ : a. a lower stem region portion being nucleotides LSI to LS6, b. a bulge region portion being nucleotides B 1 to B2, c. an upper stem region being nucleotides US 1 to US 12, d. a bulge region portion being nucleotides B3 to B6, e. a lower stem region portion being nucleotides LS7 to LS12, f. a nexus region being nucleotides N1 to N18, and g. a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ : i. a hairpin 1 region being nucleotides Hl-2, Hl-5, Hl-6, Hl-7, Hl-8, and Hl-11, ii. a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and iii. a hairpin 2 region being nucleotides H2-1 to H2-15, and h. optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: i. a 5’ end modification, j . modified nucleotides at all nucleotides in the upper stem region, k. modified nucleotides at B2 and B3 of the bulge region; l. modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region;m. modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; n. modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11 of the hairpin 1 region; and o. modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.

[0227] In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 102 or 202, optionally further comprising a 3’ tail as described herein. In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 122 or 222. In some embodiments, the gRNA conserved portion consists of the modified nucleotide sequence of SEQ ID NO: 122 or 222. In some such cases, the gRNA further comprises a spacer sequence of 17 to 20 nucleotides, such as of 20 nucleotides. In some embodiments, the gRNA comprises the modified nucleotide sequence of SEQ ID NO: 302 or 402, optionally further comprising a 3’ tail as described herein. In some cases, the 3’ tail comprises or consists of a modified uridine. In some embodiments, the gRNA comprises the modified nucleotide sequence of SEQ ID NO: 322 or 422. In some embodiments, the gRNA consists of the modified nucleotide sequence of SEQ ID NO: 322 or 422.

[0228] In other embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 117 or 217, optionally further comprising a 3’ tail as described herein. In some embodiments, the gRNA conserved portion comprises the modified nucleotide sequence of SEQ ID NO: 137 or 237. In some embodiments, the gRNA conserved portion consists of the modified nucleotide sequence of SEQ ID NO: 137 or 237. In some such cases, the gRNA further comprises a spacer sequence of 17 to 20 nucleotides, such as of 20 nucleotides. In some embodiments, the gRNA comprises the modified nucleotide sequence of SEQ ID NO: 317 or 417, optionally further comprising a 3’ tail as described herein. In some cases, the 3’ tail comprises or consists of a modified uridine. In some embodiments, the gRNA comprises the modified nucleotide sequence of SEQ ID NO: 337 or 437. In some embodiments, the gRNA consists of the modified nucleotide sequence of SEQ ID NO: 337 or 437.mRNA encoding open reading frames

[0229] As noted above, in some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding a Cas nuclease, e.g., Cas9 nuclease, as described in Table 5. In some embodiments, an mRNA comprising an ORF encoding a Cas nuclease, e.g., Cas9 nuclease, is provided, used, or administered. In some embodiments, the ORF encoding a Cas nuclease is a “modified ORF,” which is used as shorthand to indicate that the ORF is modified.

[0230] In some embodiments, the mRNA or modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, Nl-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is Nl-methyl- pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and Nl-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of Nl-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and Nl-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5- methoxyuridine.

[0231] In some embodiments, an mRNA disclosed herein comprises a 5’ cap, such as an ARCA cap, CapO, Capl, or Cap2. A 5’ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g., with respect to ARCA) linked through a 5 ’-triphosphate to the 5’ position of the first nucleotide of the 5’-to-3’ chain of the mRNA, z.e., the first cap-proximal nucleotide. In CapO, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-hydroxyl. In Capl, the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2’-methoxy and a 2’- hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33): 12025-30; and Abbas et al. (2017) Proc Natl Acad Sci USA 114(1 l):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Capl or Cap2.

[0232] A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is a cap analog comprising a 7- methylguanine 3 ’-methoxy-5’ -triphosphate linked to the 5’ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation. ARCA results in a CapO cap in which the 2’ position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis and properties of mRNAs containing the novel ‘antireverse’ cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG,” RNA 7: 1486-1495. The ARCA structure is shown below.

[0233] CleanCap™ AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap™ GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Capl structure co-transcriptionally. 3’-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively, or CleanCap AU: TriLink Biotechnologies as Cat. Nos. N-7114. The CleanCap™ AG structure is shown below.

[0234] Alternatively, a cap can be added to an RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its DI subunit, and guanine methyltransferase, provided by its D 12 subunit. As such, it can add a7-methylguanine to an RNA, so as to give CapO, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; and Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479.

[0235] In some embodiments, the mRNA further comprises a poly-adenylated (poly- A) tail. In some embodiments, the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some embodiments, the poly-A tail includes non-adenine nucleotides, i.e., is an interrupted poly-A tail. In certain embodiments, the poly-A tail is interrupted by a non-adenine nucleotide about every 40, 50, 60, 70, 80, or 90 nucleotides. In certain embodiments, the poly-A tail is interrupted by a non- adenine nucleotide about every 50 nucleotides. Exemples of the poly-A tail and further description are provided in e.g., WO2019 / 036513, the content of which is herein incorporated by reference in its entirety.

[0236] In some embodiments, the RNA encoding a Cas nuclease (e.g., mRNA) comprises a 5’ UTR, a 3’ UTR, or 5’ and 3’ UTRs. In some embodiments, the RNA (e.g., mRNA) comprises at least one UTR from Hydroxysteroid 17-Beta Dehydrogenase 4 (HSD17B4 or HSD), e.g., a 5’ UTR from HSD. In some embodiments, the RNA (e.g., mRNA) comprises at least one UTR from a globin mRNA, for example, human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA. In some embodiments, the polynucleotide (e.g., mRNA) comprises a 5’ UTR, 3’ UTR, or 5’ and 3’ UTRs from a globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the polynucleotide (e.g., mRNA) comprises a 5’ UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-al, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the polynucleotide (e.g. mRNA) comprises a 3’ UTR from bovine growth hormone, cytomegalovirus, mouse Hba-al, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the polynucleotide (e.g., mRNA) comprises 5’ and 3’ UTRs from bovine growth hormone, cytomegalovirus, mouse Hba-al, HSD, an albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).

[0237] In some embodiments, the polynucleotide (e.g., mRNA) comprises 5’ and 3’ UTRs that are from the same source, e.g., a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG.

[0238] In some embodiments, the polynucleotide (e.g., mRNA) comprises a Kozak sequence. Kozak sequences are known in the art. The Kozak sequence can affect translation initiation and the overall yield of a polypeptide translated from a nucleic acid. A Kozak sequence includes a methionine codon that can function as the start codon. A minimal Kozak sequence is NNNRUGN wherein at least one of the following is true: the first N is A or G and the second N is G. In the context of a nucleotide sequence, R means a purine (A or G). In some embodiments, the Kozak sequence is gccgccRccAUGG (SEQ ID NO: 3001) with zero mismatches or with up to one, two, three, or four mismatches to positions in lowercase.Determination of efficacy of gRNAs

[0239] In some embodiments, the efficacy of a gRNA is determined when delivered or expressed together with other components forming an RNP. In some embodiments, the gRNA is expressed together with a Cas9 nuclease, i.e., a SpyCas9 nuclease. In some embodiments, the gRNA is delivered to or expressed in a cell line that already stably expresses a Cas nuclease or nickase, i.e.., SpyCas9 nuclease or nickase. In some embodiments, the gRNA is delivered to a cell as part of an RNP. In some embodiments, the gRNA is delivered to a cell along with a mRNA encoding a Cas nuclease or nickase, i.e., SpyCas9 nuclease or nickase.

[0240] As described herein, use of a Cas nuclease and a guide RNA disclosed herein can lead to double-strand breaks in the DNA which can produce errors in the form of insertion / deletion (indel) mutations upon repair by cellular machinery. Many mutations due to indel s alter the reading frame or introduce premature stop codons and, therefore, produce a non-functional protein. In some embodiments, the efficacy of particular gRNAs is determined based on in vitro models. In some embodiments, the in vitro model is HEK293 cells stably expressing Cas9 (HEK293_Cas9). In some embodiments, the in vitro model is a primary cell line, e.g., a primary liver cell line, e.g., primary hepatocytes. In some embodiments, the primary hepatocytes are primary human hepatocytes. With respect to using primary cells, commercially available primary cells can be used to provide greater consistency between experiments. In some embodiments, the number of off-target sites at which a deletion or insertion occurs in an in vitro model (e.g., in a primary hepatocyte) is determined, e.g., by analyzing genomic DNA from cells transfected in vitro with Cas9 mRNA and the guide RNA. In some embodiments, such a determination comprises analyzing genomic DNA from the cells transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide.Exemplary procedures for such determinations are provided in the working examples in which HEK293 cells or primary hepatocytes are used.

[0241] In some embodiments, the efficacy of particular gRNAs is determined across multiple in vitro cell models for a gRNA selection process. In some embodiments, a cell line comparison of data with selected gRNAs is performed. In some embodiments, cross screening in multiple cell models is performed.

[0242] In some embodiments, the efficacy of a guide RNA is measured by percent indels of PCSK9. In some embodiments, the efficacy of a guide RNA is measured by percent indels at a PCSK9 locus. In some embodiments, the efficacy of a guide RNA is measured by percent indels of PCSK9. In some embodiments, the percent editing of PCSK9 is compared to the percent indels necessary to achieve reduction, e.g., knockdown, of the PCSK9 protein products. In some embodiments, the efficacy of a guide RNA is measured by reduced expression of PCSK9 protein. In embodiments, said reduced expression of PCSK9 protein is as measured by ELISA, e.g., as described herein.

[0243] In some embodiments, the PCSK9 protein expression is reduced in a population of cells using the methods and compositions disclosed herein. In some embodiments, the level of protein as determined, e.g., by ELISA, is reduced by at least 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, 90%, or 95% relative to a control population of unmodified cells.

[0244] An “unmodified cell” (or “unmodified cells”) refers to a control cell (or cells) of the same type of cell in an experiment or test, wherein the “unmodified” control cell has not been contacted with a, gRNA, e.g., PCSK9 gRNA. Therefore, an unmodified cell (or cells) may be a cell that has not been contacted with a guide RNA, or a cell that has been contacted with a guide RNA that does not target the genomic region being interrogated, e.g., that does not target PCSK9. The selection of an appropriate control is within the ability of those of skill in the art.

[0245] In some embodiments, the efficacy of a guide RNA is measured by the number or frequency of indels or genetic modifications at off-target sequences within the genome of the target cell type, such as a primary hepatocyte cell. In some embodiments, efficacious guide RNAs are provided which produce indels at off target sites at very low frequencies (e.g, <5%) in a cell population or relative to the frequency of indel creation at the target site. Thus, the disclosure provides for guide RNAs which do not exhibit off-target indel formation in the target cell type (e.g, a primary hepatocyte cell), or which produce a frequency of off-target indel formation of <5% in a cell population or relative to thefrequency of indel creation at the target site. In some embodiments, the disclosure provides guide RNAs which do not exhibit any off target indel formation in the target cell type (e.g., primary hepatocyte cell) as compared to a control cell. In some embodiments, guide RNAs are provided which produce indels at less than 5 validated off-target sites, e.g., as evaluated by one or more methods provided herein. In some embodiments, guide RNAs are provided which produce indels at less than or equal to 4, 3, 2, or 1 validated off-target site(s), e.g., as evaluated by one or more methods provided herein. In some embodiments, the off-target site(s) does not occur in a protein coding region in the target cell (e.g., hepatocyte) genome.

[0246] In some embodiments, the efficacy of a guide RNA is measured in vivo, e.g., in an animal or animal model having a DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA, i.e., having a DNA sequence sufficiently complementary to the targeting sequence in the guide RNA proximal to a cognate PAM for the guide and nuclease. In certain embodiments, the animal has an endogenous DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA. In certain embodiments, the animal model is a transgenic model, e.g., a mouse model having an inserted DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA, e.g., a mouse having an inserted human DNA sequence, e.g., a transgenic mouse having an inserted human PCSK9 sequence. The inserted sequence may or may not include one or more intron sequence or regulatory sequence, e.g., 3’ UTR, 5’ UTR, present in the human gene in its native context. In certain embodiments, the human DNA sequence may replace the homologous endogenous DNA sequence, e.g., the mouse PCSK9 gene is replaced by the human PCSK9 gene. In certain embodiments, the human gene is present in the mouse in the context of a human hepatocyte, e.g., a mouse with a humanized liver, e.g., as available from PheonixBio.

[0247] In some embodiments, the animal model is a rodent. In some embodiments, the rodent is a mouse or a rat. In some embodiments, the animal model is an animal expressing human PCSK9, e.g., a mouse expressing human PCSK9 from an expression construct, e.g., a viral vector, or a transgenic mouse expressing a human PCSK9. In some embodiments, the animal model is a high-fat fed, or hyperlipidimic animal, optionally an animal expressing a human PCSK9, e.g., a mouse expressing human PCSK9.

[0248] In some embodiments, detecting genome editing events, such as the formation of insertion / deletion (“indel”) mutations and insertion or homology directed repair (HDR) events in target DNA utilize linear amplification with a tagged primer and isolating the tagged amplification products (hereinafter referred to as “LAM-PCR,” or “Linear Amplification (LA)” method). In some embodiments, the efficacy of a guide RNA ismeasured by the levels of functional protein complexes comprising the expressed protein product of the gene. In some embodiments, the efficacy of a guide RNA is measured by ELISA.

[0249] Genetic modification for inhibition of target gene expression Engineered cells or population of cells comprise a genetic modification, e.g., of an endogenous nucleic acid sequence encoding PCSK9.

[0250] In some embodiments, the engineered cells or population of cells comprise a genetic modification of PCSK9 gene as assessed by sequencing, e.g., NGS, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, or 90% of cells comprise an insertion, deletion, or substitution in the endogenous PCSK9 sequence. In some embodiments, at least 50% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the endogenous PCSK9 sequence. In some embodiments, at least 80% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the endogenous PCSK9 sequence. In some embodiments, at least 85% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the endogenous PCSK9 sequence. In some embodiments, at least 90% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the endogenous PCSK9 sequence. In some embodiments, the cells in a population comprise hepatocytes in a liver. In some embodiments, PCSK9 expression is decreased by at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, or 90%, as compared to a suitable control, e.g., wherein the PCSK9 gene has not been modified. In some embodiments, expression of PCSK9 is decreased by at least 70%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein the PCSK9 gene has not been modified. In some embodiments, expression of PCSK9 is decreased by at least 75%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein the PCSK9 gene has not been modified. In some embodiments, expression of PCSK9 is decreased by at least 80%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein the PCSK9 gene has not been modified. In some embodiments, expression of PCSK9 is decreased by at least 85%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein the PCSK9 gene has not been modified. In some embodiments, expression of PCSK9 is decreased by at least 90%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein the PCSK9 gene has not been modified. In some embodiments, expression of PCSK9 is decreased by no more than 95%, as compared to asuitable control, e.g., wherein the PCSK9 gene has not been modified. Assays for PCSK9 protein and mRNA expression are known in the art.

[0251] It is understood that in some embodiments, in vivo, the level of expression may be inhibited in one, but not in all, tissues where the target gene is expressed. For example, many genes are expressed predominantly in the liver, but may also be expressed in other tissues. In certain embodiments, the level of inhibition of expression may be for a particular tissue or cell type, but not inhibition of expression systemically, e.g., inhibition of hepatic expression rather than systemic expression. In certain embodiments, surrogate markers can be used to monitor changes in expression. For example, many proteins made in the liver are secreted into circulation; therefore, the level of inhibition of expression may be determined by or correlated with a decrease in levels of the protein in the blood. In certain embodiments, inhibition of expression in the liver can result in a change in a metabolite or other biomarker in a body fluid, e.g., blood or urine. The change in the level of the metabolite can be correlated with the level of inhibition of expression. Such correlations can be useful for monitoring the level of inhibition of expression in lieu of, e.g., serial biopsies which are not practical for monitoring in human subjects, or often in animal models. The level of inhibition of expression, or absolute level of a protein in blood or serum after treatment with an agent to reduce expression of a protein in the liver have been correlated with a therapeutic outcome.

[0252] In some embodiments, the target gene is genetically modified using a guide RNA with a SpyCas9, resulting in inhibition of expression in a cell. In some embodiments, disclosed herein are cells engineered by inducing a break (e.g., double-strand break (DSB) or single-strand break (nick)) within target genes in the cells, e.g., using a guide RNA with a CRISPR / Cas system. The methods may be used in vitro, e.g., for screening guides, or in vivo, e.g., to provide a therapeutic benefit.

[0253] In some embodiments, the guide RNAs mediate a target-specific cutting by a SpyCas9 nuclease at a site described herein within a target gene. It will be appreciated that, in some embodiments, the guide RNAs comprise guide sequences that bind to, or are capable of binding to, said regions.Methods and Uses Including Therapeutic Methods and Uses of Genome Editing Agents targeted to PCSK9, VEGFA, and SERPINA1

[0254] The gRNAs and associated methods and compositions disclosed herein are useful for making genome editing therapeutic agents.

[0255] In some embodiments, the gRNAs comprising any one of the guide RNA sequences shown in Table 4 together with a SpyCas9 induce DSBs, and non-homologous end joining (NHEJ) during repair leads to a modification, e.g., a mutation, in a PCSK9 gene. In some embodiments, NHEJ leads to a deletion or insertion of a nucleotide(s), which induces a frame shift or nonsense mutation in a PCSK9 gene. In certain embodiments, gRNAs comprising guide sequences targeted to target genomic sequences are also delivered to the cell together with RNA-guided DNA nuclease such as a Cas nuclease, either together or separately, to make a genetic modification in a target genomic sequence to inhibit the expression of a full-length expression product from the target gene. In certain embodiments, the gRNAs are sgRNAs.

[0256] In some embodiments, the gRNA comprises a spacer sequence comprising a sequence at least 90% or 95% identical to the sequence of SEQ ID NO: 1. In some embodiments, the gRNA comprises a spacer sequence comprising 18 or 19 contiguous nucleotides of SEQ ID NO: 1. In some embodiments, the gRNA comprises a spacer sequence comprising the sequence of SEQ ID NO: 1. Exemplary sgRNAs targeting PCSK9 are disclosed in Table 4A.

[0257] In some embodiments, the gRNAs comprising any one of the guide RNA sequences shown in Table 4 together with a SpyCas9 induce DSBs, and non-homologous end joining (NHEJ) during repair leads to a modification, e.g., a mutation, in a VEGFA or SEPPINA1 gene. In some embodiments, NHEJ leads to a deletion or insertion of a nucleotide(s), which induces a frame shift or nonsense mutation in a VEGFA or SERPINA1 gene. In certain embodiments, gRNAs comprising guide sequences targeted to target genomic sequences are also delivered to the cell together with RNA-guided DNA nuclease such as a Cas nuclease, either together or separately, to make a genetic modification in a target genomic sequence to inhibit the expression of a full-length expression product from the target gene. In certain embodiments, the gRNAs are sgRNAs.

[0258] In some embodiments, the gRNA comprises a spacer sequence comprising a sequence at least 90% or 95% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the gRNA comprises a spacer sequence comprising 18 or 19 contiguous nucleotides of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the gRNA comprises a spacer sequence comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. Exemplary sgRNAs targeting VEGFA or SERPINA1 are disclosed in Table 4B.Table 4A. Exemplary sgRNA sequences targeting PCSK9Table 4B. Exemplary sgRNA sequences targeting VEGFA or SERPINA1

[0259] In some embodiments, the guide RNA comprises a modified nucleotide sequence selected from SEQ ID NOs: 602, 608, 609, 607, 603, or 604. In some embodiments, the guide RNA comprises a modified nucleotide sequence of SEQ ID NO: 602. In some embodiments, the guide RNA comprises a modified nucleotide sequence of SEQ ID NO: 607. In some embodiments, the guide RNA comprises a modified nucleotide sequence of SEQ ID NO: 603. In some embodiments, the guide RNA comprises a modified nucleotide sequence of SEQ ID NO: 604.

[0260] In some embodiments, the guide RNAs, compositions, and formulations are used to produce a cell in vivo, e.g, liver cell, e.g, a hepatocyte with a genetic modification in PCSK9 gene. In some embodiments, the cell is in a subject.

[0261] In some embodiments, the subject is mammalian. In some embodiments, the subject is human. In some embodiments, the subject is a non-human primate.

[0262] In some embodiments, the subject has or is at risk of having a PCSK9-related disease or condition. A " / YA -associated disease or condition", or a “ / YA -related disease or condition” as used herein, is intended to include any disease or condition associated with the PCSK9 gene or protein expression or activity. Such a disease or condition may be caused, for example, by excess production of the PCSK9 protein, by PCSK9 gene mutations, by abnormal cleavage of the PCSK9 protein, by abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9- associated diseases include lipidemias, e.g., hyperlipidemias, and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases. In some embodiments, a / YA -related disease or condition is selected from the group consisting of a cardiovascular disease or a chronic liver injury. In some embodiments, a / Y A' -related disease or condition includes, but is not limited to, hypercholesterolemia (e.g., total blood cholesterol levels > 190mg / dl, or LDL-cholesterol levels > 100 mg / dl), familial hypercholesterolemia (FH), autosomal dominant hypercholesterolemia (ADH), autosomal recessive hypercholesterolemia (ARH), hyperlipidemia, hypertriglyceridemia, coronary artery disease, stroke, myocardial infarction, obesity, xanthoma, atherosclerosis, aortic stenosis, liver steatosis, high blood pressure, type 2 diabetes, and insulin resistance.

[0263] Familial hypercholesterolemia (FH) is characterized by severely elevated LDL cholesterol (LDL-C) levels (e.g., over 190 mg / dL in adults, or over 160 mg / dL in children) that lead to atherosclerotic plaque deposition in the coronary arteries and proximal aorta at an early age, leading to an increased risk for cardiovascular disease, which may manifest as angina, myocardial infarction, or stroke. FH is a genetic disease that is passed on through family, and is caused by a pathogenic mutation in one of three genes (APOB, LDLR, and PCSK9). Patients can be heterozygous or homozygous for the mutation.

[0264] Autosomal dominant hypercholesterolemia (ADH) and autosomal recessive hypercholesterolemia (ARH) are other forms of hypercholesterolemia, also characterized by excessive blood cholesterol levels (Cohen, J. C, (2003) Curr. Opin. Lipidol. 14, 121-127). ADH is due to defects in LDL uptake by the liver, which may be caused by LDLR mutations that prevent LDL uptake, or by mutations in the protein on LDL, apolipoprotein B, which is responsible for LDL binding to LDLR. ARH is caused by mutations in the ARH protein that are necessary for endocytosis of the LDLR-LDL complex via its interaction with clathrin. Itis understood that subjects with a EGS' -associated disease may be treated with one or more additional therapeutic agents within the standard of care for treatment of lipid disorders, or conditions associated with lipid disorders, e.g., cardiovascular disease, e.g., high blood pressure; type 2 diabetes, insulin resistance. In certain embodiments, the subject is treated with the additional agent until a reduction of serum PCSK9 level is observed. In certain embodiments, the subject is treated with an additional agent until a change is observed in a sign or symptom associated with the / YA -associated disease, e.g., a reduction of blood pressure prior to discontinuation of treatment with one or more agents to reduce hypertension; a normalization of blood sugar or blood sugar regulation prior to discontinuation of treatment with one or more agents to normalize blood sugar or blood sugar regulation. In some embodiments, the subject is treated with one or more PCSK9 inhibitors. In some embodiments, the subject is treated with one or more anti-PCSK9 monoclonal antibodies. In some embodiments, the subject is treated with evolocumab. In some embodiments, the subject is treated with alirocumab. In some embodiments, the subject is treated with vutrisiran.

[0265] Examples of additional therapeutic agents include those known to treat a lipid disorder, such as hypercholesterolemia, atherosclerosis or dyslipidemia. For example, a gRNA featured in the invention can be administered with, e.g., an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a glycoprotein Ilb / IIIa inhibitor, aspirin or an aspirinlike compound, an IB AT inhibitor, a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin, pravastatin, simvastatin, lovastatin, fluvastatin, cerivastatin, rosuvastatin, and pitivastatin. Exemplary fibrates include, e.g., bezafibrate, clofibrate, fenofibrate, gemfibrozil, and ciprofibrate. Exemplary bile acid sequestrants include, e.g., cholestyramine, colestipol, and colesevelam. Exemplary niacin therapies include, e.g., immediate release and extended release formulation. Exemplary antiplatelet agents include, e.g., aspirin, clopidogrel, and ticlopidine. Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril and enalapril. Exemplary acyl CoA cholesterol acetyltransferase (AC AT) inhibitors include, e.g., avasimibe and eflucimibe. Exemplarycholesterol absorption inhibitors include, e.g., ezetimibe and pamaqueside. Exemplary CETP inhibitors include, e.g., Torcetrapib, JTT-705, and CETi-I. Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide, R-103757, and CP- 346086.

[0266] Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu / Banyu), Btg-511 (British Technology Group), BARI- 1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD- 7806 (AstraZeneca). Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar, netoglitazone. Exemplary Glycoprotein Ilb / IIIa inhibitors include, e.g., roxifiban, gantofiban, and cromafiban. The anti- atherosclerotic agent BO- 653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin are also appropriate for administering in combination with a gRNA featured in the invention. Exemplary combination therapies suitable for administration with a gRNA targeting PCSK9 include, e.g., advicor, amlodipine / atorvastatin, and ezetimibe / simvastatin. Agents for treating hypercholesterolemia, and suitable for administration in combination with a gRNA targeting PCSK9 include, e.g., lovastatin, amlodipine besylate, atorvastatin, rosuvastatin, fluvastatin, niacin, pravastatin, fenofibrate, ezetimibe, simvastatin, colesevelam, and ezetimibe.

[0267] In certain embodiments, methods comprise instructing an end user, e.g., a healthcare provider, a subject, to administer an additional agent, such as that provided above, in conjunction with administration of a gRNA provided herein. In certain embodiments, an additional agent, i.e., one or more additional agents, is administered in conjunction with, e.g., before, at the time of, or after administration of the gRNA, for example, until a desired clinical outcome is reached, e.g., reduction of blood pressure or serum cholesterol or lipid; normalization of blood sugar.

[0268] In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The method includes administering to the patient a gRNA in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without substantially lowering HDL levels.

[0269] Genetic predisposition plays a role in the development of target gene associated diseases, e.g., hyperlipidemia. However, the lack of functional evidence for most variants detected during the molecular screening of patients with clinical familial hypercholesterolemia (FH) can make the definitive diagnosis difficult (see, e.g., Di Costanzo et al. (2021) I Clin Lipidol, 15:822-831). Therefore, a patient in need of a gRNA can beidentified by taking a family history, or, for example, screening for one or more genetic markers or variants, typically in conjunction with, or prompted by, signs for hyperlipidemia. Examples of genes involved in hyperlipidemia include but are not limited to, e.g., LDL receptor (LDLR), the apoliproteins (ApoAl, ApoB, ApoE, and the like), Cholesteryl ester transfer protein (CETP), Lipoprotein lipase (LPL), hepatic lipase (LIPC), Endothelial lipase (EL), Lecithinxholesteryl acyltransferase (LCAT). It is expected that population based genomic studies, e.g., UK Biobank, will further define genetic markers associated with familial hypercholesterolemia and other hyperlipidemias.

[0270] A healthcare provider, such as a doctor, nurse, or geneticist can take a family history before prescribing or administering a gRNA agent of the invention. In addition, a test may be performed to determine a genotype or phenotype. For example, a DNA test may be performed on a sample from the patient, e.g., a blood sample, to identify the PCSK9 genotype or phenotype before a PCSK9 gRNA is administered to the patient. Variants in P( 'SK9.jboth pathogenic and benign, can be found, for example in the NCBI SNP database at www.ncbi.nlm.nih. gov / snp / ?LinkName=gene_snp&from_uid=255738. In another embodiment, a test is performed to identify a related genotype or phenotype, e.g., an LDLR genotype. Examples of genetic variants with the LDLR gene can be found in the art, e.g., in the following publications which are incorporated by reference: Costanza et al (2005) Am J Epidemiol. 15; 161(8):714-24; Yamada et al. (2008) J Med Genet. Jan;45(l):22-8, Epub 2007 Aug 31; and Boes et al (2009) Exp. Gerontol 44: 136-160, Epub 2008 Nov 17.Compositions, kits, and systems

[0271] Compositions comprising any of the gRNAs described herein and a carrier, excipient, diluent, or the like are encompassed. In some instances, the excipient or diluent is inert. In some instances, the excipient or diluent is not inert. In some embodiments, a pharmaceutical formulation is provided comprising any of the gRNAs described herein and a pharmaceutically acceptable carrier, excipient, diluent, or the like. In some embodiments, the pharmaceutical formulation further comprises an LNP. In some embodiments, the pharmaceutical formulation further comprises a Cas9 protein or an mRNA encoding a Cas9 protein. In some embodiments, the pharmaceutical formulation comprises any one or more of the gRNAs, an LNP, and a Cas9 protein or mRNA encoding a Cas9 protein. In some embodiments, the pharmaceutical formulation is non-pyrogenic.

[0272] Also provided are kits comprising one or more gRNAs, compositions, or pharmaceutical formulations described herein. In some embodiments, a kit further comprisesone or more of a solvent, solution, buffer, each separate from the composition or pharmaceutical formulation, instructions, or desiccant.

[0273] Also provided are systems comprising one or more gRNAs, compositions, or pharmaceutical formulations described herein. In some embodiments, the system is for inducing a double stranded break in a PCSK9 sequence in a genome wherein the system includes a sgRNA provided herein and a SpyCas9 cleavase. In some embodiments, the system is for inducing indel formation in a PCSK9 sequence in a genome wherein the system includes a sgRNA provided herein and a SpyCas9 cleavase.Compositions comprising a SpyCas9 polypeptide or an mRNA encoding a SpyCas9 polypeptide

[0274] In some embodiments, compositions or pharmaceutical formulations are provided comprising at least one gRNA described herein and a SpyCas9 polypeptide or a nucleic acid (e.g., an mRNA) encoding a SpyCas9 polypeptide. In some embodiments, the gRNA together with a Cas protein or nucleic acid (e.g., mRNA) encoding Cas9 polypeptide is called a Cas9 RNP. In some embodiments, compositions are provided comprising at least one gRNA and a nuclease or an mRNA encoding a spyCas9. In some embodiments, the Cas induces a double strand break in target DNA. Equivalents of spyCas9 protein are encompassed by the embodiments described herein.

[0275] As used herein, Cas9 encompasses modified and variant versions of Cas9. Modified versions having one catalytic domain, either RuvC or HNH, that is inactive are termed “nickases.” Nickases cut only one strand on the target DNA, thus creating a singlestrand break. A single-strand break may also be known as a “nick.” In some embodiments, the compositions and methods comprise nickases. In some embodiments, the compositions and methods comprise a nickase, such as a nickase Cas9, that induces a nick rather than a double strand break in the target DNA.

[0276] In some embodiments, the Cas9 may be modified to contain only one functional nuclease domain. For example, the Cas9 may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase Cas9 is used having a RuvC domain with reduced activity. In some embodiments, a nickase Cas9 is used having an inactive RuvC domain. In some embodiments, a nickase Cas9 is used having an HNH domain with reduced activity. In some embodiments, a nickase Cas9 is used having an inactive HNH domain.

[0277] In some embodiments, a conserved amino acid within a Cas9 is substituted to reduce or alter nuclease activity. In some embodiments, a Cas9 protein may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). In some embodiments, the Cas9 protein may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863 A, H983 A, and D986A (based on the spyCas9 protein).

[0278] In some embodiments, the RNP complex described herein comprises a nickase or an mRNA encoding a nickase and a pair of gRNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the gRNAs direct the nickase to a target sequence and introduce a double stranded break (DSB) by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate gRNAs that are selected to be in close proximity to produce a double nick in the target DNA.

[0279] In some embodiments, chimeric Cas proteins are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fokl. In some embodiments, a Cas protein may be a modified nuclease.

[0280] In some embodiments, the Cas protein comprises a fusion protein comprising a catalytically inactive Cas nuclease (e.g., Cas9) linked to a heterologous functional domain (see, e.g., WO2014152432). In some embodiments, the catalytically inactive Cas9 is from S. pyogenes. In some embodiments, the catalytically inactive Cas9 comprises mutations that inactivate the Cas9. In some embodiments, the heterologous functional domain is a domain that modifies gene expression, histones, or DNA. In some embodiments, the heterologous functional domain is a transcriptional activation domain or a transcriptional repressor domain. In some embodiments, the nuclease is a catalytically inactive Cas nuclease, such as dCas9.

[0281] In some embodiments, the target sequence may be adjacent to a PAM. In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4, nucleotides of the 3' end of the target sequence. The length and the sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus or a particular PAM sequence for a specific Cas9 protein or Cas9 ortholog, including those disclosed in Figure 1 of Ran et al., Nature 520: 186-191 (2015). In some embodiments, the PAM may comprise 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NAG, NGA, NGAG, NGCG, NNGRRT, TTN, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, and NNNNGATT (wherein N is defined as any nucleotide, and W is defined as either A or T, and R is defined as either A or G). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be NNAAAAW.

[0282] In some embodiments, the heterologous functional domain may facilitate transport of the Cas nuclease into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the Cas nuclease may be fused with 1-5 NLS(s). In some embodiments, the Cas nuclease may be fused with one NLS. Where one NLS is used, the NLS may be fused at the N-terminus or the C-terminus of the Cas nuclease sequence. It may also be inserted within the Cas nuclease sequence. In other embodiments, the Cas nuclease may be fused with more than one NLS. In some embodiments, the Cas nuclease may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the Cas nuclease may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the Cas nuclease is fused to two NLS sequences (e.g., SV40) at the carboxy terminus. In some embodiments, the Cas nuclease may be fused with two NLSs, one at the N-terminus and one at the C-terminus. In some embodiments, the Cas nuclease may be fused with 3 NLSs. In some embodiments, the Cas nuclease may be fused with no NLS. In some embodiments, the NLS may be a monopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 1001) or PKKKRRV (SEQ ID NO: 1002). In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 1003). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 1001) NLS may be fused at the C-terminus of the Cas nuclease. One or more linkers are optionally included at the fusion site.

[0283] In some embodiments, the heterologous functional domain may be an effector domain. In some embodiments, the heterologous functional domain is a deaminase domain, such as a cytidine deaminase or an adenine deaminase. In certain embodiments, the heterologous functional domain is a C to T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase. In certain embodiments, the APOBEC deaminase is an APOBEC3 A deaminase. In certain embodiments, the APOBEC3 A deaminase is an 7 / . sapiens APOBEC3A deaminase comprising the amino acid sequence of SEQ ID NO: 1084. In certain embodiments, the composition disclosed herein comprises auracil glycosylase inhibitor (UGI) or an mRNA encoding a UGI. In some embodimnets, the UGI comprises the amino acid sequence of SEQ ID NO: 1088. In some embodiments, the mRNA encoding a UGI comprises an open reading frame (ORF) comprising the sequence SEQ ID NO: 1087. In certain embodiments, the composition disclosed herein comprises a base editor (BE) or an mRNA encoding a base editor, wherein the base editor comprises the SpyCas9 nickase and the deaminase domain. In certain embodiments, the base editor comprises the amino acid sequence of SEQ ID NO: 1086. In some embodiments, the mRNA encoding the base editor comprises an open reading frame (ORF) comprising the sequence of SEQ ID NO: 1085. Additional examples and further description of such base editor and deaminase that may be used herein for the heterologous functional domain are provided in e.g., WO2022 / 125968, the content of which is herein incorporated by reference in its entirety.

[0284] In some embodiments, the heterologous functional domain comprises a polymerase domain.

[0285] In certain embodiments, a heterologous functional domain can include a Cas9 DNA targeting unit (see, e.g., WO2016106338, the content of which is herein incorporated by reference in its entirety). In certain embodiments, a heterologous functional domain can include a polymerase, e.g., a DNA polymerase (see, e.g., W02019051097, the content of which is herein incorporated by reference in its entirety).In some embodiments, a nucleic acid (e.g., mRNA) comprising an ORF encoding a Cas nuclease is used which has one or more of the following features. In some embodiments, the ORF encoding the S. pyogenes Cas9, is as described in WO2019067910 or W02020198641 or WO2021119275, incorporated herein by reference.

[0286] In any of the foregoing embodiments, the nucleic acid may be an mRNA.Exemplary sequences

[0287] In some embodiments, the ORF encoding the Cas9 nuclease provided herein comprises a sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 1102-1122, 1125, 1126, or 1129-1146. In some embodiments, the mRNA encoding the nuclease provided herein comprises a sequence with at least 90% identity to any one of SEQ ID NOs: 1085, 1093, 1096, 1099-1127, or 1129- 1146. In some embodiments, the mRNA comprises the sequence of any one of SEQ ID NOs: 1085, 1093, 1096, 1099-1127, or 1129-1146. In some embodiments, any of the foregoing levels of identity is at least 95%, 98%, 99%, or 100%.

[0288] In some embodiments, the mRNA comprises a sequence with at least 90% identity to any one of SEQ ID NOs: 1101, 1123, 1124, or 1127, wherein the sequence comprises an ORF encoding a Cas9 nuclease. In some embodiments, the mRNA comprises a sequence with at least 90% identity to any one of SEQ ID NOs: 1101, 1123, 1124, or 1127, wherein the sequence comprises an ORF encoding a Cas9 nuclease, wherein the first three nucleotides of SEQ ID NOs: 1101, 1123, 1124, or 1127 are omitted. In some embodiments, the mRNA comprises a sequence with at least 90% identity to any one of SEQ ID NOs: 1101, 1123, 1124, or 1127, wherein the sequence comprises an ORF encoding a Cas9 nuclease, wherein the first three nucleotides of SEQ ID NOs: 1101, 1123, 1124, or 1127 are omitted and / or the ORF coding sequence contained within SEQ ID NO: 1101, 1123, 1124, or 1127 is substituted with the coding sequence of any one of SEQ ID NOs: 1102-1122, 1125, 1126, or 1129-1146. In some embodiments, any of the foregoing levels of identity is at least 95%, 98%, 99%, or 100%.Methods of Gene Modulation

[0289] In some embodiments, any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein is for use in preparing a medicament for treating or preventing a disease or disorder in a subject.

[0290] In some embodiments, the present disclosure provides a method of treating a disease or disorder in subject comprising administering any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein.

[0291] In some embodiments, the present disclosure provides a method or use of modifying a target DNA comprising, administering or delivering any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein.

[0292] In some embodiments, the present disclosure provides a method or use for modulation of a target gene comprising, administering or delivering any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein. In some embodiments, the modulation is editing of the target gene. In some embodiments, the modulation is a change in expression of the protein encoded by the target gene.

[0293] In some embodiments, the method or use results in genome editing. In some embodiments, the method or use results in a double-stranded break within the target gene. In some embodiments, the method or use results in formation of indel mutations during non- homologous end joining of the DSB. In some embodiments, the method or use results in an insertion or deletion of nucleotides in a target gene. In some embodiments, the insertion ordeletion of nucleotides in a target gene leads to a frameshift mutation or premature stop codon that results in a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in a target gene leads to a knockdown of target gene expression. In some embodiments, the method or use comprises homology directed repair of a DSB. In some embodiments, the method or use further comprises delivering to the cell a template, wherein at least a part of the template incorporates into a target DNA at or near a double strand break site induced by the nuclease.

[0294] In some embodiments, the method or use results in a nick or single-stranded break within the target gene by the Spy Cas9 nuclease, wherein the nuclease is provided with, optionally fused to a deaminase, such as a cytidine deaminase or an adenine deaminase, to induce a point mutation or base change, e.g., a deamination.

[0295] In some embodiments, the method or use results in a nick or single-stranded break within the target gene by the Spy Cas9 nuclease, wherein the nuclease is provided with, optionally fused to a polymerase. In certain embodiments, the method or use further includes a template to direct a change in the genome sequence, e.g., base change, insertion, or deletion.

[0296] In some embodiments, the method or use results in gene modulation. In some embodiments, the method or use results in decreased expression of the protein encoded by the target gene.

[0297] The efficacy of gRNAs can be tested in vitro and in vivo. In some embodiments, the present disclosure provides one or more of the gRNAs, compositions, or pharmaceutical formulations described herein, wherein the gRNA results in gene modulation when provided to a cell together with Cas9 or mRNA encoding Cas9. In some embodiments, the efficacy of gRNA can be measured in vitro or in vivo.

[0298] In some embodiments, the activity of a Cas RNP comprising a gRNA is compared to the activity of a Cas RNP comprising an unmodified sgRNA or a reference sgRNA lacking modifications present in the sgRNA, such as a control sgRNA with a modification pattern provided in W02018107028 or WO2021119275.

[0299] In some embodiments, the efficiency of a gRNA in decreasing target protein expression is determined by measuring the amount of target protein.

[0300] In some embodiments, the efficiency of editing with specific gRNAs is determined by the editing present at the target location in the genome following delivery of Cas9 and the gRNA. In some embodiments, the efficiency of editing with specific gRNAs is measured by next-generation sequencing. In some embodiments, the editing percentage of thetarget region of interest is determined. In some embodiments, the total number of sequence reads with insertions or deletions of nucleotides into the target region of interest over the total number of sequence reads is measured following delivery of a gRNA and Cas9.

[0301] In some embodiments, the efficiency of editing with specific gRNAs is measured by the presence of insertions or deletions of nucleotides introduced by successful genome editing. In some embodiments, activity of a Cas9 and gRNAs is tested in biochemical assays. In some embodiments, activity of a Cas9 and gRNAs is tested in a cell- free cleavage assay.

[0302] In some embodiments, the activity of modified gRNAs is measured after in vivo dosing of LNPs comprising modified gRNAs and Cas protein or mRNA encoding Cas protein.

[0303] In some embodiments, in vivo efficacy of a gRNA or composition provided herein is determined by editing efficacy measured in DNA extracted from tissue (e.g., liver tissue) after administration of gRNA and Cas9.

[0304] In some embodiments, activation of the subject’s immune response is measured by serum concentrations of cytokine(s) following in vivo dosing of sgRNA together with Cas9 mRNA or protein (e.g., formulated in a LNP). In some embodiments, the cytokine is interferon-alpha (IFN-alpha), interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and / or tumor necrosis factor alpha (TNF-alpha).

[0305] In some embodiments, administration of Cas RNP or Cas9 mRNA together with the modified gRNA produces lower serum concentration(s) of immune cytokines compared to administration of unmodified sgRNA. In some embodiments, the present disclosure provides a method of reducing a subject’s serum concentration of immune cytokines comprising, administering any one of the gRNAs disclosed herein, wherein the gRNA produces a lower concentration of immune cytokines in a subject’s serum as compared to a control gRNA that is not similarly modified.LNP delivery of gRNA / composition

[0306] Lipid nanoparticles (LNPs) are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of the gRNAs, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.

[0307] In some embodiments, the present disclosure provides a method for delivering any one of the gRNAs or compositions disclosed herein to a subject, wherein the gRNA isassociated with an LNP. In some embodiments, the gRNA / LNP is also associated with a Cas9 or an mRNA encoding Cas9.

[0308] In some embodiments, the present disclosure provides a composition comprising any one of the gRNAs disclosed and an LNP. In some embodiments, the composition further comprises a Cas9 or an mRNA encoding Cas9.

[0309] In some embodiments, a method for delivering any one of the gRNAs disclosed herein in vivo is provided, wherein the gRNA is associated with an LNP. In some embodiments, the gRNA is not associated with an LNP.

[0310] In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for treating a disease or disorder.

[0311] In some embodiments, the nucleic acid compositions described herein, comprising a gRNA and a nucleic acid described herein encoding a Cas nuclease e.g. Cas9, are formulated in or systemically administered via a lipid nanoparticle; see e.g., WO2017173054A1 entitled “LIPID NANOPARTICLE FORMULATIONS FOR CRISPR / CAS COMPONENTS,” and WO2019067992A1 entitled “FORMULATIONS,” the contents of which, in particular the LNP compositions disclosed therein, are hereby incorporated by reference in their entirety. Lipid nanoparticles (LNPs) known to those of skill in the art to be capable of delivering therapeutic RNAs to subjects may be utilized with the guide RNAs described herein and the nucleic acid encoding a Cas nuclease.

[0312] Compositions comprising LNPs may include two active substances, a guideRNA and an RNA encoding a Cas nuclease, e.g. a Cas9 nuclease such as a Spy. Cas9 nuclease, together with a lipid component comprising an ionizable lipid. By lipid nanoparticle is meant a particle that comprises a plurality of (i.e. more than one) lipid molecules physically associated with each other by intermolecular forces.Ionizable Lipids

[0313] Lipid compositions for delivery of CRISPR / Cas mRNA and guide RNA components to a liver cell may comprise Lipid A, which is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate. Lipid A can be depicted as:

[0314] Lipid A may be synthesized according to W02015 / 095340 (e.g., pp. 84-86).Additional Lipids

[0315] “Neutral lipids” suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-l,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn- glycero-3 -phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2- diarachidoyl-sn-glycero-3 -phosphocholine (DBPC), l-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3 -phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).

[0316] “Helper lipids” include steroids, sterols, and alkyl resorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5- heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol.

[0317] “Stealth lipids” are lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood), and a stealth lipid may be a PEG lipid. Stealth lipids mayassist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition of the disclosure include, but are not Typically, the PEG lipid comprises a lipid moiety and a polymer moiety based on PEG. PEG lipids known in the art are contemplated, including lipids comprising a “PEG- 2K,” also termed “PEG 2000,” which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (I), wherein n is 45, meaning that the number averaged degree of polymerization comprises about 45 subunits. However, other PEG embodiments known in the art may be used.

[0318] In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-di stearoylglycerol (PEG-DSPE) (catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG- dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- [omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol)ether), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG2k-DMPE), or l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene glycol -2000 (PEG2k-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #8801200 from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and l,2-distearyloxypropyl-3-amine-N- [methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG.

[0319] In some embodiments, the PEG lipid includes a glycerol group. In some embodiments, the PEG lipid includes a dimyristoylglycerol (DMG) group. In some embodiments, the PEG lipid comprises PEG2k. In some embodiments, the PEG lipid is a PEG-DMG. In some embodiments, the PEG lipid is a PEG2k-DMG. In some embodiments, the PEG lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In someembodiments, the PEG2k-DMG is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0320] The LNP composition may comprise a lipid component and an RNA component that includes a Cas nuclease mRNA (e.g. a Cas9 mRNA, such as a Spy. Cas9 mRNA), and a gRNA. In some embodiments, an LNP composition includes an mRNA encoding a Cas9 nuclease and a gRNA as the RNA component. In certain embodiments, an LNP composition may comprise the RNA component, Lipid A, a helper lipid, a neutral lipid, and a stealth lipid. In certain LNP compositions, the helper lipid is cholesterol. In certain compositions, the neutral lipid is DSPC. In additional embodiments, the stealth lipid is PEG2k-DMG.

[0321] In certain embodiments, lipid compositions are described according to the respective molar ratios of the component lipids in the formulation. Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the formulation. In one embodiment, the mol-% of the ionizable lipid such as Lipid A is about 40 mol-%- 60 mol-%, optionally about 50 mol.

[0322] In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, is from about 5 mol-%- 15 mol-%, optionally about 9 mol-%.

[0323] In one embodiment, the mol-% of the helper lipid is from about 20 mol-% -60 mol-%, optionally about 25 mol-% -55 mol-%, optionally, about 30 mol-% to 40 mol-%.

[0324] In one embodiment, the mol-% of the PEG lipid is about 1 mol-%- 10 mol-%, optionally about 2.5 mol-% - 4 mol-%, optionally about 3 mol-%.

[0325] In certain embodiments, the cargo includes a nucleic acid (e.g., mRNA) encoding a Cas9 nuclease and a sgRNA. In some embodiments, the ionizable lipid is Lipid A. In some embodiments, an LNP composition comprises an ionizable lipid (e.g. Lipid A), a neutral lipid, a helper lipid, and a PEG lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In specific embodiments, PEG lipid is PEG2k-DMG. In some embodiments, an LNP composition may comprise a Lipid A, a helper lipid, a neutral lipid, and a PEG lipid. In additional embodiments, an LNP composition comprises Lipid A, cholesterol, DSPC, and PEG2k-DMG.

[0326] Embodiments of the present disclosure also provide lipid compositions described according to the molar ratio between the positively charged ionizable groups of the ionizable lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the ratio N / P. In some embodiments, an LNP composition may comprise a lipid component that comprises anionizable lipid, a helper lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component, wherein the N / P ratio is about 3 to 10. In some embodiments, the N / P ratio is about 5-7, optionally the N / P ratio is about 6.

[0327] In some embodiments, the RNA component comprises an mRNA, such as a nucleic acid disclosed herein, encoding a Cas9 nuclease, e.g., a Spy Cas9 nuclease, mRNA described herein, and a sgRNA described herein. In any of the foregoing embodiments, the sgRNA is a chemically modified sgRNA described herein.

[0328] In certain embodiments, the LNP compositions include a Cas9 nuclease mRNA (such as a Spy Cas9 mRNA) described herein and a sgRNA described herein. In certain embodiments, the LNP composition includes a ratio of gRNA to Cas9 nuclease mRNA, such as a Spy Cas9 nuclease mRNA from about 10: 1- 1 : 10, e.g., about 1 : 1, 1 :2, or 1 :3.

[0329] In some embodiments, LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, e.g., 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceutically acceptable buffer, e.g., for in vivo administration of LNPs, may be used.

[0330] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, flow rates, junction size, junction geometry, junction shape, tube diameter, solutions, or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, e.g., via dialysis, tangential flow filtration, or chromatography. The LNPs may be composed of 4 lipids including Lipid A; DSPC; cholesterol; and DMG-PEG2k. In some embodiments, the LNP is suspended and formulated in an aqueous buffer of 50 mM Tris, 45 mM NaCl, and 5% (w / v) sucrose, pH 7.4.

[0331] Dynamic Light Scattering (“DLS”) can be used to characterize the poly dispersity index (“pdi”) and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (around the mean particle size) in a population, with a perfectly uniform population having a PDI of zero.

[0332] In some embodiments, LNPs disclosed herein have a size of 50 to 100 nm. In some embodiments, the LNPs have a size of 85 to 90 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample isdiluted in phosphate buffered saline (PBS) so that the count rate is approximately 200-400 kcts. The data are presented as a weighted-average of the intensity measure.

[0333] In some embodiments, LNPs associated with the gRNAs disclosed herein and mRNA encoding a Spy Cas9 nuclease disclosed herein are for use in preparing a medicament for treating a PCSK9 associated disease. In some embodiments, LNPs associated with the gRNAs disclosed herein and mRNA encoding a Spy Cas9 nuclease disclosed herein are for use in preparing a medicament for reducing PCSK9 in circulation, for example in a subject having a PCSK9 associated disease, e.g., as detected in blood or serum derived therefrom. In some embodiments, LNPs associated with the gRNAs disclosed herein and mRNA encoding a Spy Cas9 nuclease disclosed herein are for use in preparing a medicament for promoting a double stranded break in a PCSK9 genomic sequence, e.g., a human PCSK9 genomic sequence. In some embodiments, LNPs associated with the gRNAs disclosed herein and mRNA encoding a Spy Cas9 nuclease disclosed herein are for use in preparing a medicament for promoting indel formation in a PCSK9 genomic sequence, e.g., a human PCSK9 genomic sequence.

[0334] In some embodiments, the LNP comprises a lipid component and the lipid component comprises, consists essentially of, or consists of: about 50 mol-% ionizable lipid such as Lipid A; about 9 mol-% neutral lipid such as DSPC; about 3 mol-% of a stealth lipid such as a PEG lipid, such as PEG2k-DMG, and the remainder of the lipid component is helper lipid such as cholesterol, wherein the N / P ratio of the LNP composition is about 6. In some embodiments, the ionizable lipid is Lipid A. In some embodiments, the neutral lipid is DSPC. In some embodiments, the stealth lipid is a PEG lipid. In some embodiments, the stealth lipid is a PEG2k-DMG. In some embodiments, the helper lipid is cholesterol. In some embodiments, the LNP comprises a lipid component and the lipid component comprises: about 50 mol-% Lipid A; about 9 mol-% DSPC; about 3 mol-% of PEG2k-DMG, and the remainder of the lipid component is cholesterol wherein the N / P ratio of the LNP composition is about 6.

[0335] Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivery of any one of the gRNAs disclosed herein. In some embodiments, electroporation may be used to deliver any one of the gRNAs disclosed herein and Cas9 or an mRNA encoding Cas9.

[0336] In some embodiments, the present disclosure provides a method for delivering any one of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNA is associatedwith an LNP or not associated with an LNP. In some embodiments, the gRNA / LNP or gRNA is also associated with a Cas9 or an mRNA encoding Cas9.Exemplary Embodiments

[0337] Exemplary embodiments of the present disclosure include, but are not limited to, the following:Embodiment 1 : A guide RNA (gRNA) comprising: a spacer sequence of 17-20 nucleotides in length, and a conserved portion comprising the nucleotide sequence of SEQ ID NO: 709 or 710, wherein i . nucleotides 5 ’ -GUUUUAGAGCUAGAAAUAGC AAGUUAAAAU-3 ’ or 5’-GUUUUAGACGUAGAAAUACGAAGUUAAAAU-3’ (SEQ ID NO: 3002 or 3003) constitute a repeat / antirepeat (R / AR) region, wherein nucleotides 1-6 (LS1-LS6) and 25-30 (LS7-LS12) of the R / AR constitute a lower stem (LS) region, nucleotides 7-8 (B1-B2) and 21-24 (B3-B6) of the R / AR constitute a bulge region; and nucleotides 9-20 (US 1 -US 12) of the R / AR constitute an upper stem (US) region; ii. nucleotides 5’-AAGGCUAGUCCGUUAUCA-3’ (SEQ ID NO: 3004) constitute a nexus region (N1-N18); iii. nucleotides 5’-CGAAAG-3’ constitute a hairpin 1 (Hl) region (from 5’ to 3’, Hl-2, Hl-5 to Hl-8, and Hl-11); iv. nucleotides 5’-GCACCGAGUCGGUGC-3’ (SEQ ID NO: 3007) constitute a hairpin 2 (H2) region H2-1 to H2-15); and v. a G nucleotide between Hl region and H2 region constitutes nucleotide n; wherein the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: i. the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-0-Me at LS2, LS3, LS4, LS5; ii. the bulge region comprising nucleotides not modified with 2’-0-Me at B5 and B6; iii. the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9,US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; iv. the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, and N14; v. the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl- 5; vi. the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; and vii. the n is a modified or unmodified nucleotide; wherein the gRNA comprises a 5’ end modification.Embodiment 2: The gRNA of embodiment 1, wherein the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: i. the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, and LS6; ii. the bulge region comprising nucleotides not modified with 2’-O-Me at B5 and B6; iii. the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; iv. the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16; v. the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl- 5;vi. the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; and vii. the n is a modified or unmodified nucleotide.Embodiment 3: The gRNA of embodiment 1, wherein the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: i. the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, LS6, and LS9; ii. the bulge region comprising nucleotides not modified with 2’-O-Me at B5 and B6; iii. the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; iv. the nexus region comprising modified nucleotides at Nl, N2, N4, N7, Ni l, N12, N16, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, N14, and N16; v. the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl- 5; vi. the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-11, H2-12, H2-13, H2-14, and H2-15; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; and vii. the n is a modified or unmodified nucleotide.Embodiment 4: The gRNA of any one of claims 1-3, wherein the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1.Embodiment 5: The gRNA of any one of embodiments 1-4, wherein the lower stem region comprises unmodified nucleotides at LS2, LS3, LS4, and LS5, optionally at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1.Embodiment 6: The gRNA of any one of embodiments 1-5, wherein the lower stem region comprises unmodified nucleotides at LS7, LS9, and LSI 1.Embodiment 7: The gRNA of any one of embodiments 1-6, wherein the bulge region comprises modified nucleotides at B2 and B3.Embodiment 8: The gRNA of any one of embodiments 1-7, wherein the bulge region comprises unmodified nucleotides at B5 and B6, optionally at Bl, B4, B5, and B6. Embodiment 9: The gRNA of any one of embodiments 1-8, wherein the modified nucleotides of the upper stem are 2’-0-Me modified nucleotides.Embodiment 10: The gRNA of any one of embodiments 1-9, wherein the nexus region comprises unmodified nucleotides at N6, N8, N9, N10, N13, N14, and N18, optionally at N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18.Embodiment 11 : The gRNA of any one of embodiments 1-10, wherein the nexus region comprises unmodified nucleotides at N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18. Embodiment 12: The gRNA of any one of embodiments 1-11, wherein the hairpin 1 region comprises an unmodified nucleotide at Hl -5.Embodiment 13: The gRNA of any one of embodiments 1-11, wherein the hairpin 2 region comprises unmodified nucleotides at H2-8, H2-9, and H2-10.Embodiment 14: The gRNA of any one of embodiments 1-13, further comprising a 3’ tail.Embodiment 15: The gRNA of any one of embodiments 1-14, wherein the gRNA comprises a 3’ end modification.Embodiment 16: The gRNA of any one of embodiments 1-15, wherein the spacer sequence is 20 nucleotides in length.Embodiment 17: The gRNA of embodiment 16, wherein the spacer sequence comprises modified nucleotides at SI, S2, and S3; and nucleotides not modified with 2’-O-Me at S5, S6, S7, S12, S15, S16, and S19.Embodiment 18: The gRNA of embodiment 16, wherein the spacer sequence comprises modified nucleotides at SI, S2, and S3; and nucleotides not modified with 2’-O-Me at S5, S6, S7, S8, S12, S15, S16, and S19.Embodiment 19: The gRNA of embodiment 16-18, wherein the spacer sequence further comprises modified nucleotides at S9 and S13.Embodiment 20: The gRNA of embodiment 16-20, wherein the spacer sequence comprises modified nucleotides at SI, S2, S3, S4, S7, S8, S9, S10, SI 1, S13, S14, S17 and S18. Embodiment 21 : The gRNA of any one of embodiments 1-20, wherein at least one modified nucleotide comprises a modified nucleotide that stabilizes A-form helix structure, optionallyI l lwherein each modified nucleotide comprising a 2’-ribose modification comprises a modified nucleotide that stabilizes A-form helix structure.Embodiment 22: The gRNA of any one of embodiments 1-21, wherein each modified nucleotide comprises a modification independently selected from 2’-O-methyl (2’-0-Me) modified nucleotide, a 2’ -O-(2 -methoxy ethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, bicyclic ribose analog, a locked nucleic acid (LNA), ethylene- bridged nucleic acids (ENA), bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), and a phosphorothioate modification.Embodiment 23: The gRNA of any one of embodiments 1-22, wherein each modified nucleotide comprises a modification independently selected from 2’-O-methyl (2’-0-Me) modified nucleotide, a 2’ -O-(2 -methoxy ethyl) (2’-O-moe) modified nucleotide, a 2’-fhioro (2’-F) modified nucleotide, bicyclic ribose analog, a locked nucleic acid (LNA), ethylene- bridged nucleic acids (ENA), bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), and a phosphorothioate modification.Embodiment 24: The gRNA of any one of embodiments 1-23, wherein each modified nucleotide is independently selected from 2’-O-methyl (2’-0-Me) modified nucleotide, a 2’- O-(2 -methoxy ethyl) (2’-O-moe) modified nucleotide, a 2’ -fluoro (2’-F) modified nucleotide, and a phosphorothioate modified nucleotide; optionally wherein each modified nucleotide comprises a 2’-0-Me modified nucleotide.Embodiment 25: The gRNA of any one of embodiments 1-24 wherein at least one modified nucleotide comprising a 2’ -ribose modification further comprises a phosphorothioate modified nucleotide, optionally a 2’-0-Me modified nucleotide and a phophosphorothioate modified nucleotide.Embodiment 26: The gRNA of any one of embodiments 1-25, wherein each nucleotide not modified with 2’-0-Me is independently selected from a 2’-fluoro (2’-F) modified nucleotide and an unmodified nucleotide.Embodiment 27: The gRNA of any one of embodiments 1-26, wherein the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, 110, 111, and 112.Embodiment 28: The gRNA of any one of embodiments 1-27, wherein the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, 210, 211, and 212.Embodiment 29: The gRNA of any one of embodiments 1-28, wherein the gRNA comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, and 210.Embodiment 30: The gRNA of any one of embodiments 1-29, wherein the gRNA comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, and 204.Embodiment 31: The gRNA of any one of embodiments 27-30, wherein each modified nucleotide in the H2 region is independently selected from 2’-O-methyl (2’-O-Me) modified nucleotide, a 2’ -O-(2 -meth oxy ethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, bicyclic ribose analog, a locked nucleic acid (LNA), ethylene-bridged nucleic acids (ENA), bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), and a phosphorothioate modified nucleotide.Embodiment 32: The gRNA of any one of embodiments 27-31, wherein each modified nucleotide in the H2 region is independently selected from 2’-O-methyl (2’-O-Me) modified nucleotide, a 2’ -O-(2 -meth oxy ethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, and a phosphorothioate modified nucleotide.Embodiment 33: The gRNA of any one of embodiments 27-32, wherein at least one modified nucleotide in the H2 region comprises a 2’ -ribose modification and a phosphorothioate modified nucleotide, optionally a 2’-0-Me modified nucleotide and a phosphosphorothioate modified nucleotide.Embodiment 34: The gRNA of any one of embodiments 1-33, wherein the modified nucleotides of the H2 region comprises a 2’-0-Me modified nucleotide.Embodiment 35: The gRNA of any one of embodiments 1-34, wherein each modified nucleotide in the H2 region comprises a 2’-0-Me modified nucleotide.Embodiment 36: The gRNA of any one of embodiments 27-35, wherein the gRNA comprises a 3’ tail.Embodiment 37: The gRNA of any one of embodiments 14-36, wherein the 3’ tail is 1-4 nucleotides, optionally 1 nucleotide in length.Embodiment 38: The gRNA of embodiment 34 or 35, wherein the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, optionally wherein the modified nucleotide is a 2’-0-Me modified nucleotide or a phosphorothioate modified nucleotide.Embodiment 39: The gRNA of embodiment 37, wherein the 3’ tail is fully modified.Embodiment 40: The gRNA of any one of embodiments 34-37, wherein the 3’ tail comprises a terminal 2’-O-Me modified uridine nucleotide wherein the terminal 2’-O-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage. Embodiment 41 : The gRNA of any one of embodiments 1-13 and 15-33, wherein the gRNA does not comprise a 3’ tail.Embodiment 42: The gRNA of any one of embodiments 1-41, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are modified nucleotides.Embodiment 43: The gRNA of any one of embodiments 1-42, wherein the first three nucleotides at the 5’ end of the 5’ terminus are modified nucleotides.Embodiment 44: The gRNA of any one of embodiments 1-43, wherein the first two, three, or four nucleotides at the 5’ end of the 5’ terminus are 2’-0-Me modified nucleotides.Embodiment 45: The gRNA of any one of embodiments 1-44, wherein the first two, three, or four nucleotides at the 5’ end of the 5’ terminus are linked with phosphorothioate (PS) bonds. Embodiment 46: The gRNA of any one of embodiments 1-45, wherein the first two, three, or four nucleotides at the 5’ end of the 5’ terminus are 2’-0-Me modified nucleotides, further wherein the first two, three, or four nucleotides at the 5’ end of the 5’ terminus are linked with phosphorothioate (PS) bonds.Embodiment 47: The gRNA of any one of embodiments 1-46, wherein the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are modified nucleotides.Embodiment 48: The gRNA of any one of embodiments 1-47, wherein the last three nucleotides at the 3’ end of the 3’ terminus are modified nucleotides.Embodiment 49: The gRNA of any one of embodiments 1-48, wherein the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are 2’-0-Me modified nucleotides.Embodiment 50: The gRNA of any one of embodiments 1-49, wherein the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are linked with phosphorothioate (PS) bonds. Embodiment 51 : The gRNA of any one of embodiments 1-50, wherein the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are 2’-0-Me modified nucleotides, further wherein the last two, three, or four nucleotides at the 3’ end of the 3’ terminus are linked with phosphorothioate (PS) bonds.Embodiment 52: The gRNA of any one of embodiments 1-51, wherein the spacer comprises a modification pattern selected from:SEQ ID NOs: 302-304 or 402-404;SEQ ID NOs: 307 and 407; andSEQ ID NOs: 308, 408, 309, 409, 310, 410, 311, 411, 312, and 412.Embodiment 53: The gRNA of embodiment 52, wherein the spacer comprises a modification pattern selected fromSEQ ID NOs: 302-304 and 402-404;SEQ ID NOs: 307 and 407; andSEQ ID NOs: 308, 408, 309, 409, 310, 410.Embodiment 54: The gRNA of embodiment 53, wherein the spacer comprises a modification pattern selected fromSEQ ID NOs: 302-304 and 402-404; and SEQ ID NOs: 307 and 407.Embodiment 55: The gRNA of any one of embodiments 1-54, wherein the modified nucleotide sequence of the conserved portion is selected from:SEQ ID NOs: 102 and 202;SEQ ID NOs: 107 and 207;SEQ ID NOs: 103, 203, 104, and 204; andSEQ ID NOs: 108, 208, 109, 209, 110, 210, 111, 211, 112, and 212.Embodiment 56: The gRNA of embodiment 55, wherein the modified nucleotide sequence of the conserved portion is selected from:SEQ ID NOs: 102 and 202;SEQ ID NOs: 107 and 207;SEQ ID NOs: 103, 203, 104, and 204; andSEQ ID NOs: 108, 208, 109, 209, 110, 210.Embodiment 57: The gRNA of embodiment 56, wherein the modified nucleotide sequence of the conserved portion is selected from:SEQ ID NOs: 102 and 202;SEQ ID NOs: 107 and 207; andSEQ ID NOs: 103, 203, 104, and 204.Embodiment 58: The gRNA of any one of embodiments 1-57, wherein the modified nucleotide sequence is selected from:SEQ ID NOs: 302 and 402;SEQ ID NOs: 307 and 407;SEQ ID NOs: 303, 403, 304, and 404; andSEQ ID NOs: 308, 408, 309, 409, 310, 410, 311, 411, 312, and 412.Embodiment 59: The gRNA of embodiment 58, wherein the modified nucleotide sequence is selected from: a) SEQ ID NOs: 302 and 402; b) SEQ ID NOs: 307 and 407; c) SEQ ID NOs: 303, 403, 304, and 404; and d) SEQ ID NOs: 308, 408, 309, 409, 310, 410.Embodiment 60: The gRNA of embodiment 59, wherein the modified nucleotide sequence is selected from: a) SEQ ID NOs: 302 and 402; b) SEQ ID NOs: 307 and 407; c) SEQ ID NOs: 303, 403, 304, and 404.Embodiment 61 : The gRNA of any one of embodiments 1-60, wherein the nucleotide sequence of the spacer comprises SEQ ID NO: 1.Embodiment 62: A lipid nanoparticle (LNP) composition comprising the gRNA of any one of embodiments 1-61.Embodiment 63: A composition comprising the gRNA of any one of embodiments 1-61 associated with a lipid nanoparticle (LNP).Embodiment 64: A composition comprising the gRNA of any one of embodiments A1-A52, or the composition of embodiment Bl or B2, further comprising a S. pyogenes Cas9 (SpyCas9) nuclease or a nucleic acid which encodes a SpyCas9 nuclease.Embodiment 65: The composition of embodiment 64, wherein the nuclease is a cleavase.Embodiment 66: The composition of embodiment 65, wherein the nuclease is a nickase or a dCas.Embodiment 67: The composition of any one of embodiments 64-66, wherein the nuclease comprises a modified PAM interacting domain.Embodiment 68: The composition of any one of embodiments 64-67, wherein the nuclease comprises a heterologous functional domain.Embodiment 69: The composition of embodiment 68, wherein the heterologous functional domain comprises a nuclear localization signal (NLS).Embodiment 70: The composition of embodiment 68 or 69, wherein the heterologous functional domain comprises a deaminase domain.Embodiment 71 : The composition of embodiment 70, wherein the composition comprises a uracil glycosylase inhibitor (UGI) or an mRNA encoding a UGI.Embodiment 72: The composition of embodiment 68 or 69, wherein the heterologous functional domain comprises a polymerase domain.Embodiment 73: The composition of any one of embodiments 64-72, comprising an mRNA which encodes the nuclease.Embodiment 74: The composition of embodiment 73, wherein the mRNA comprises the sequence of any one of SEQ ID NOs: 1085, 1093, 1096, 1099-1127 or 1129-1146.Embodiment 75: A system comprising the gRNA of any one of embodiments 1-61 and an S. pyogenes Cas9 nuclease or an mRNA which encodes an S. pyogenes Cas9 nuclease of any one of embodiments 64-74.Embodiment 76: A kit comprising the gRNA of any one of embodiments 1-61 and an S. pyogenes Cas9 nuclease or an mRNA which encodes an S. pyogenes Cas9 nuclease of any one of embodiments 64-74.Embodiment 77: A pharmaceutical formulation comprising the gRNA of any one of embodiments 1-61 or the composition of any one of embodiments 62-74 and a pharmaceutically acceptable carrier.Embodiment 78: A method of modifying a target DNA comprising, delivering a S. pyogenes Cas9 (SpyCas9) nuclease or a nucleic acid encoding a SpyCas9 nuclease, and any one or more of the following to a cell: i. the gRNA of any one of embodiments 1-61; ii. the composition of any one of embodiments 62-74; iii. the system of embodiment 75; iv. the kit of embodiment 76; v. the pharmaceutical formulation of embodiment 77.Embodiment 79: The method of embodiment 78, wherein the method results in an insertion or deletion in a gene.Embodiment 80: The method of embodiment 78 or 79, further comprising delivering to the cell a template, wherein at least a part of the template incorporates into a target DNA at or near a double strand break site induced by the SpyCas9 nuclease.Embodiment 81 : The gRNA of any one of embodiments 1-61, the composition of embodiments 62-74, the system of embodiment 75, the kit of embodiment 76, or thepharmaceutical formulation of embodiment 77 for use in preparing a medicament for treating a disease or disorder.Embodiment 82: Use of the gRNA of any one of embodiments 1-61, the composition of embodiments 62-74, the system of embodiment 75, the kit of embodiment 76, or the pharmaceutical formulation of embodiment 77 in the manufacture of a medicament for treating a disease or disorder.Embodiment 83: A guide RNA comprising a conserved portion and a spacer sequence, wherein the conserved portion is selected from: a conserved portion of any one of embodiments 1-61; and mGUUUfUAGmAmGmCmUmAmGmAmAmAmUmAmGmCmAmA GUfUmAfAmAfAmUAmAmGmGmCmUmAGUmCmCGUfUAmUm CAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmC mGmAmGmUmCmGmGmUmGmC (SEQ ID NO: 620); and the spacer sequence is selected from: a spacer sequence comprising a sequence at least 95% or 90% identical to the sequence of SEQ ID NO: 1; a spacer sequence comprising a sequence identical to 18 or 19 contiguous nucleotides of the sequence of SEQ ID NO: 1; or a spacer sequence comprising the sequence of SEQ ID NO: 1.Embodiment 84: The guide of embodiment 83, comprising the sequence of SEQ ID NO: 1. Embodiment 85: The guide RNA of embodiment 83 or 84, wherein the guide RNA comprises a modified nucleotide sequence selected from:SEQ ID NOs: 102, 202, 302, 402, or 602;SEQ ID NOs: 107, 207, 307, 407, or 607;SEQ ID NOs: 103, 203, 303, 403, or 603; orSEQ ID NOs: 104, 204, 304, 404, or 604.Embodiment 86: The guide RNA of embodiment 85, wherein the guide RNA comprises a modified nucleotide sequence selected from:SEQ ID NOs: 102, 202, 302, or 402; or SEQ ID NOs: 107, 207, 307, or 407.Embodiment 87: The guide RNA of embodiment 83 comprising the modified nucleotide sequence of any one of SEQ ID NOs: 602, 607, 603, 604, 621 or 618.Embodiment 88: The gRNA of embodiment 83 or 84, further comprising a 3’ tail, optionally wherein the 3’ tail is 1-4 nucleotides in length.Embodiment 89: The gRNA of any one of embodiments 83, 84, or 88, wherein the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, optionally wherein the modified nucleotide is a 2’-0-Me modified nucleotide or a phosphorothioate modified nucleotide, optionally wherein the 3’ tail comprises a terminal 2’- O-Me modified uridine nucleotide wherein the terminal 2’-0-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage, and further optionally the 3’ tail is fully modified.Embodiment 90: A composition comprising the guide RNA of any one of embodiments 83- 89.Embodiment 91 : The composition of embodiment 90, further comprising a S. pyogenes Cas9 or a nucleic acid comprising an open reading frame (ORF) encoding a SpyCas9 nuclease, wherein the nuclease has double-stranded endonuclease activity.Embodiment 92: The composition of embodiment 91, wherein the nucleic acid is an mRNA. Embodiment 93: The composition of embodiment 91 or 92, wherein the S. pyogenes Cas9 comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 1089, 1092, and 1095.Embodiment 94: The composition of any one of embodiments 90-93, wherein the guide RNA comprises the sequence of SEQ ID NO: 1 and the SpyCas9 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1089.Embodiment 95: The composition of any one of embodiments 90-93, wherein the guide RNA comprises the sequence of SEQ ID NO: 1 and the SpyCas9 comprises the amino acid sequence of SEQ ID NO: 1089.Embodiment 96: The composition of embodiment 92, wherein the mRNA comprises an ORF encoding a SpyCas9 having at least 90% identity to any one of SEQ ID NOs: 1091 and 1094. Embodiment 97: The composition of embodiment 96, wherein the ORF encoding the amino acid sequence has at least 95% identity, optionally at least 98% identity to any one of SEQ ID NOs: 1091 and 1094.Embodiment 98: The composition of embodiment 97, wherein the ORF encoding the amino acid sequence comprises SEQ ID NO: 1091 or 1094.Embodiment 99: The composition of any one of embodiments 90-98, wherein the guide RNA comprises the sequence of SEQ ID NO: 1 and wherein an ORF encoding a SpyCas9 comprises at least 95% identity to SEQ ID NO: 1091.Embodiment 100: The composition of any one of embodiments 90-99, wherein the ORF is a modified ORF.Embodiment 101 : The composition of any one of embodiments 97-100, wherein the guide RNA comprises the nucleotide sequence of SEQ ID NO: 709 or 710.Embodiment 102: The composition of any one of embodiments 97-101, wherein the guide RNA comprises the modified nucleotide sequence of any one of SEQ ID NOs: 602, 607, 603, and 604.Embodiment 103: The composition of any one of embodiments 90-102, further comprising a pharmaceutical excipient.Embodiment 104: The composition of any one of embodiments 90-103, wherein the guide RNA is associated with a lipid nanoparticle (LNP).Embodiment 105: The composition of embodiment 104, wherein the LNP comprises a cationic lipid.Embodiment 106: The composition of embodiment 105, wherein the cationic lipid is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate.Embodiment 107: The composition of any one of embodiments 104-106, wherein the LNP comprises a helper lipid.Embodiment 108: The composition of embodiment 107, wherein the helper lipid is cholesterol.Embodiment 109: The composition of any one of embodiments 104-108, wherein the LNP comprises a neutral lipid.Embodiment 110: The composition of embodiment 109, wherein the neutral lipid is 1,2- di stearoyl -sn-gly cero-3 -phosphocholine (D SPC) .Embodiment 111 : The composition of any one of embodiments 104-110, wherein the LNP comprises a stealth lipid.Embodiment 112: The composition of embodiment 111, wherein the stealth lipid is 1,2- dimyristoyl-rac-gly cero-3 -methoxypolyethylene glycol -2000 (PEG2k-DMG).Embodiment 113: The composition of embodiment 104, wherein the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-di enoate, DSPC, cholesterol, and PEG2k-DMG.Embodiment 114: A pharmaceutical composition comprising the guide RNA of any one of embodiments 83-89 or the composition of any one of embodiments 90-113.Embodiment 115: A pharmaceutical composition comprising, or use of, the guide RNA of any one of embodiments 83-89 or the composition of any one of embodiments 90-113 for inducing a double-strand break within a PCSK9 gene in a cell or reducing expression of a PCSK9 gene in a cell.Embodiment 116: The pharmaceutical composition or use of embodiment 115, wherein the cell is a liver cell.Embodiment 117: The pharmaceutical composition or use of embodiment 116, wherein the cell is in a subject.Embodiment 118: A pharmaceutical composition comprising, or use of, the guide RNA of any one of embodiments 83-89 or the composition of any one of embodiments 90-113 for treating a subject having a PCSK9 related disease.Embodiment 119: A method of inducing a double-strand break within a PCSK9 gene in a cell or reducing expression of a PCSK9 protein in a cell comprising contacting a cell with the guide RNA of any one of embodiments 83-89 and a SpyCas9 nuclease or a nucleic acid encoding a SpyCas9 nuclease wherein the nuclease has double-stranded endonuclease activity, or the composition of any one of embodiments 90-113.Embodiment 120: A method of modifying a genomic locus in a human liver cell, the method comprising contacting a human liver cell with the guide RNA of any one of embodiments 83- 89 and a SpyCas9 nuclease or a nucleic acid encoding a SpyCas9 nuclease wherein the nuclease has double-stranded endonuclease activity, or the composition of any one of embodiments 90-113.Embodiment 121 : The method of embodiment 120, wherein the method is performed in vivo. Embodiment 122: The pharmaceutical composition, method, or cell of any one of embodiments 116, 117, and 119-121, wherein the liver cell or cell is a hepatocyte.Embodiment 123: The pharmaceutical composition, method, or cell of embodiment 122, wherein the cell is in a subject with a PCSK9 related disease.Embodiment 124: A method of treating a PCSK9 related disease in a subject, the method comprising administering to the subject the guide RNA of any one of embodiments 83-89 and a SpyCas9 nuclease or a nucleic acid encoding a SpyCas9 nuclease wherein the nuclease has double-stranded endonuclease activity, or the composition of any one of embodiments 90- 113, or the pharmaceutical composition of embodiment 114.Embodiment 125: The pharmaceutical composition for use or method of any one of embodiments 115-119 and 121-124, further comprising determining the PCSK9 protein level in a subject blood or serum sample.Embodiment 126: Use of the guide RNA of any one of embodiments 83-89 or the composition of any one of embodiments 90-113, or the pharmaceutical composition of embodiment 114 in the preparation of a medicament for practicing the method of any one of embodiments 119 and 121-125.Embodiment 127: A system for editing PCSK9 gene, comprising the guide RNA of any one of embodiments 83-89 and an S. pyogenes Cas9 nuclease or an mRNA which encodes an S. pyogenes Cas9 nuclease of any one of embodiments 94-96.Embodiment 128: A system for treating a PCSK9 related disease in a subject, comprising the guide RNA of any one of embodiments 83-89 and an S. pyogenes Cas9 nuclease or an mRNA which encodes an S. pyogenes Cas9 nuclease of any one of embodiments 94-96..Embodiment 129: A kit comprising the guide RNA of any one of embodiments 83-89 and an S. pyogenes Cas9 nuclease or an mRNA which encodes an S. pyogenes Cas9 nuclease wherein the nuclease has double-stranded endonuclease activity, the composition of any one of embodiments 90-113, or the pharmaceutical composition of embodiment 114.Embodiment 130: A kit for use or for practicing the method of any one of embodiments 119 and 121-125.

[0338] Additional exemplary embodiments are included below.Embodiment Al. A guide RNA (gRNA) comprising, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’: a. a lower stem region portion being nucleotides LSI to LS6, b. a bulge region portion being nucleotides B 1 to B2, c. an upper stem region being nucleotides US1 to US 12, optionally wherein the upper stem region further comprises up to 8 additional nucleotides, d. a bulge region portion being nucleotides B3 to B6,e. a lower stem region portion being nucleotides LS7 to LS12, f. a nexus region being nucleotides N1 to N18, and g. a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ : i. a hairpin 1 region being nucleotides Hl-1 to Hl-12, optionally wherein the hairpin 1 region lacks up to 6 nucleotides of Hl-1 to Hl-12, ii. a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and iii. a hairpin 2 region being nucleotides H2-1 to H2-15; and h. optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: i. a 5’ end modification, j. modified nucleotides at 10, 11, or all nucleotides in the upper stem region, k. 3 or more modified nucleotides in the hairpin 2 region, wherein the gRNA further comprises one or more additional modifications selected from: l. modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; m. modified nucleotides at B2 and B3 of the bulge region; n. modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; o. modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11 of the hairpin 1 region, wherein the hairpin 1 region consists of nucleotides Hl-2, Hl -5, Hl-6, Hl-7, Hl-8 and Hl-11; p. modified nucleotides at all nucleotides of the hairpin 1 region, wherein the hairpin 1 region comprises Hl-1 to Hl-12; and q. modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.Embodiment A2. The gRNA of embodiment Al, wherein the conserved portion comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 400, SEQ ID NO: 709 or SEQ ID NO: 710.Embodiment A3. The gRNA of embodiment A2, wherein the conserved portion comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 701, or 702.Embodiment A4. The gRNA of embodiment A3, wherein the gRNA comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 801, 802, 809, or 810.Embodiment A5. The gRNA of any one of embodiments A1-A4, wherein the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: a. the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, optionally further comprising nucleotides not modified with 2’-O-Me at LS6 and / or LS9; b. the bulge region comprising nucleotides not modified with 2’-O-Me at B5 and B6; c. the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; d. the nexus region comprising modified nucleotides at N1 , N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, and N14, optionally further comprising a nucleotide not modified with 2’-O-Me at N16; e. the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl- 7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl-5; f. the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2- 3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15, optionally further comprising a modified nucleotide at H2-11; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; and g. the n is a modified or unmodified nucleotide.Embodiment A6. The gRNA of any one of embodiments A1-A5, wherein the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12; and nucleotides not modified with 2’-O-Me at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1.Embodiment A7. The gRNA of any one of embodiments A1-A6, wherein the lower stem region comprises unmodified nucleotides at LS2, LS3, LS4, and LS5, optionally at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1.Embodiment A8. The gRNA of any one of embodiments A1-A7, wherein the lower stem region comprises unmodified nucleotides at LS7, LS9, and LSI 1.Embodiment A9. The gRNA of any one of embodiments A1-A8, wherein the bulge region comprises modified nucleotides at B2 and B3.Embodiment A10. The gRNA of any one of embodiments A1-A9, wherein the bulge region comprises unmodified nucleotides at B5 and B6, optionally at Bl, B4, B5, and B6.Embodiment Al 1. The gRNA of any one of embodiments A1-A10, wherein the nexus region comprises modified nucleotides atNl, N2, N4, N7, Ni l, N12, and N17.Embodiment A12. The gRNA of any one of embodiments Al-Al l, wherein the nexus region comprises unmodified nucleotides atN6, N8, N9, N10, N13, N14, and N18, optionally at N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18.Embodiment A13. The gRNA of any one of embodiments A1-A12, wherein the hairpin 1 region comprises modified nucleotides at Hl -2, Hl -6, Hl -7, Hl -8, and Hl-11.Embodiment A14. The gRNA of any one of embodiments A1-A13, wherein the hairpin 1 region comprises an unmodified nucleotide at Hl -5.Embodiment A15. The gRNA of any one of embodiments A1-A14, wherein the hairpin 2 region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-25.Embodiment Al 6. The gRNA of any one of embodiments Al -Al 5, wherein the hairpin 2 region comprises unmodified nucleotides at H2-8, H2-9, and H2-10.Embodiment A17. A guide RNA (gRNA) comprising, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’: a. a lower stem region portion being nucleotides LSI to LS6, b. a bulge region portion being nucleotides B 1 to B2, c. an upper stem region being nucleotides US 1 to US 12, d. a bulge region portion being nucleotides B3 to B6, e. a lower stem region portion being nucleotides LS7 to LS12, f. a nexus region being nucleotides N1 to N18, and g. a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ : iv. a hairpin 1 region being nucleotides Hl -2, Hl -5, Hl -6, Hl -7, Hl -8, and Hl-11,v. a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and vi. a hairpin 2 region being nucleotides H2-1 to H2-15, and h. optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: i. a 5’ end modification, j . modified nucleotides at all nucleotides in the upper stem region, k. modified nucleotides at B2 and B3 of the bulge region; l. modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; m. modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; n. modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11 of the hairpin 1 region; and o. modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.Embodiment A18. The gRNA of embodiment A17, wherein: a. the lower stem region comprises unmodified nucleotides at LS2, LS3, LS4, and LS5, optionally at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1; b. the bulge region comprises unmodified nucleotides at B5 and B6, optionally at B1, B4, B5, and B6; c. the nexus region comprises unmodified nucleotides atN6, N8, N9, N10, N13, N14, and N18, optionally at N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18; d. the hairpin 1 region comprises an unmodified nucleotide at Hl-5; and e. the hairpin 2 region comprises unmodified nucleotides at H2-8, H2-9, and H2- 10.Embodiment Al 9. The gRNA of any one of embodiments Al -Al 8, wherein the spacer sequence comprises one or more modified nucleotide.Embodiment A20. The gRNA of any one of embodiments Al -Al 9, wherein the spacer sequence comprises one or more phosphorothioate (PS) modifications.Embodiment A21. The gRNA of any one of embodiments A1-A20, wherein the spacer sequence comprises one or more modified nucleotide selected from a 2’-O-methyl (2’-O-Me) modified nucleotide or a 2’-fluoro (2’-F) modified nucleotide.Embodiment A22. The gRNA of any one of embodiments A1-A21, wherein the spacer sequence is 20 nucleotides in length.Embodiment A23. The gRNA of any one of embodiments A1-A22, wherein the spacer sequence comprises 2’-O-Me modified nucleotides at each of the first three 5’ terminal nucleotides (SI, S2, and S3).Embodiment A24. The gRNA of any one of embodiments A1-A23, wherein the spacer sequence comprises one or more phosphorothioate (PS) linkages between the first four 5’ terminal nucleotides.Embodiment A25. The gRNA of any one of embodiments A22-A24, wherein the spacer sequence comprises modified nucleotides at SI, S2, and S3; and nucleotides not modified with 2’-0-Me at S5, S6, S7, S12, S15, S16, and S19; optionally comprising nucleotides not modified with 2’-0-Me at S5, S6, S7, S8, S12, S15, S16, and S19.Embodiment A26. The gRNA of any one of embodiments A23-A25, wherein the spacer sequence further comprises modified nucleotides at S9 and S13.Embodiment A27. The gRNA of embodiment A26, wherein the spacer sequence comprises modified nucleotides at SI, S2, S3, S4, S7, S8, S9, S10, Si l, S13, S14, S17 and S18.Embodiment A28. The gRNA of any one of embodiments A1-A27, wherein at least one modified nucleotide comprises a modified nucleotide that stabilizes A-form helix structure, optionally wherein each modified nucleotide comprising a 2’ -ribose modification comprises a modified nucleotide that stabilizes A-form helix structure.Embodiment A29. The gRNA of any one of embodiments A1-A28, wherein each modified nucleotide comprises a modification independently selected from a 2’-O- methyl (2’-O-Me) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a bicyclic ribose analog, a locked nucleic acid (LNA), ethylene-bridged nucleic acids (ENA), a bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), and a phosphorothioate (PS) modification.Embodiment A30. The gRNA of any one of embodiments A1-A29, wherein at least one modified nucleotide comprising a 2’ -ribose modification further comprises a phosphorothioate modified nucleotide, optionally a 2’-O-Me modified nucleotide and a phophosphorothioate modified nucleotide.Embodiment A31. The gRNA of any one of embodiments A1-A30, wherein each nucleotide not modified with 2’-0-Me is independently selected from a 2’-fluoro (2’- F) modified nucleotide and an unmodified nucleotide.Embodiment A32. The gRNA of any one of embodiments Al -A31, wherein the modified nucleotides of the upper stem region are 2’-0-Me modified nucleotides.Embodiment A33. The gRNA of any one of embodiments A1-A32, wherein the modified nucleotides of the lower stem region, bulge, nexus region, hairpin 1 region, or hairpin 2 region, and optionally each of the lower stem region, bulge, nexus region, hairpin 1 region, and hairpin 2 region are 2’-0-Me modified nucleotides.Embodiment A34. The gRNA of any one of embodiments A1-A33, wherein the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, 110, 111, 112, 122, 127, 123, 124, 128, 129, 130, 131, and 132.Embodiment A35. The gRNA of any one of embodiments A1-A33, wherein the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, 210, 211, 212, 122, 127, 123, 124 128, 129, 130, 131, and 132.Embodiment A36. The gRNA of any one of embodiments A1-A33, wherein the conserved portion comprises a modified nucleotide sequence of SEQ ID NO: 102, 202, 122, or 222.Embodiment A37. The gRNA of any one of embodiments A1-A36, wherein the gRNA comprises a 3’ end modification.Embodiment A38. The gRNA of embodiment A37, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are modified nucleotides.Embodiment A39. The gRNA of embodiment A37 or A38, wherein the last three nucleotides at the 3’ end of the gRNA are modified nucleotides.Embodiment A40. The gRNA of any one of embodiments A37-A39, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are 2’-O-Me modified nucleotides.Embodiment A41. The gRNA of any one of embodiments A37-A40, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are linked with phosphorothioate (PS) bonds.Embodiment A42. The gRNA of any one of embodiments A37-A41, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are 2’-O-Me modifiednucleotides, further wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are linked with phosphorothioate (PS) bonds.Embodiment A43. The gRNA of any one of embodiments A1-A42, further comprising a 3’ tail, optionally wherein the 3’ tail is 1, 2, 3, or 4 nucleotides, optionally 1 nucleotide in length.Embodiment A44. The gRNA of embodiment A43, wherein the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, optionally wherein the modified nucleotide is a 2’-0-Me modified nucleotide or a phosphorothioate modified nucleotide.Embodiment A45. The gRNA of embodiment A44, wherein the 3’ tail is fully modified.Embodiment A46. The gRNA of any one of embodiments 43-45, wherein the 3’ tail comprises a terminal 2’-0-Me modified uridine nucleotide wherein the terminal 2’-O- Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage.Embodiment A47. The gRNA of any one of embodiments A1-A2 or A5-A42, wherein the gRNA does not comprise a 3’ tail.Embodiment A48. The gRNA of any one of embodiments A1-A47, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are modified nucleotides.Embodiment A49. The gRNA of embodiment A48, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are 2’-0-Me modified nucleotides.Embodiment A50. The gRNA of embodiment A48 or A49, wherein the first three nucleotides at the 5’ end of the gRNA are modified nucleotides.Embodiment A51. The gRNA of any one of embodiments A48-A50, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are linked with phosphorothioate (PS) bonds.Embodiment A52. The gRNA of any one of embodiments A48-A51, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are 2’-0-Me modified nucleotides, further wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are linked with phosphorothioate (PS) bonds.Embodiment A53. A gRNA comprising a modified nucoleotide sequence selected from: a. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 302, 402, 322, and 422;b. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 307, 407, 327, and 427; c. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 303, 403, 304, 404, 323, 423, 324, and 424; and d. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 308, 408, 309, 409, 310, 410, 311, 411, 312, 412, 328, 428, 329, 429, 330, 430, 331, 431, 332, and 432.Embodiment A54. A gRNA comprising a modified nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 302, 402, 322, and 422.Embodiment A55. A gRNA comprising a modified nucleotide sequence selected from SEQ ID Nos: 302 and 402, and further comprising a 3’ tail, optionally wherein the 3’ tail is 1, 2, 3, or 4, nucleotides, and optionally wherein the 3’ tail comprises a terminal 2’-0-Me modified uridine nucleotide wherein the terminal 2’-0-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage.Embodiment A56. A gRNA comprising a modified nucleotide sequence selected from SEQ ID NOs: 322 and 422.Embodiment A57. The gRNA of any one of embodiments A1-A56, further comprising a spacer comprising the nucleotide sequence of SEQ ID NO: 1.Embodiment A58. A gRNA consisting of the modified nucleotide sequence of SEQ ID NOs: 322 or 422.Embodiment A59. A gRNA consisting of the modified nucleotide sequence of SEQ ID NO: 322 or 422, wherein the nucleotide sequence of the first 20 nucleotides is the sequence of SEQ ID NO: 1.Embodiment A60. A gRNA comprising the modified nucleotide sequence of any one of SEQ ID NOs: 601-619.Embodiment A61. A gRNA consisting of the modified nucleotide sequence of any one of SEQ ID NOs: 601-619.Embodiment A62. A gRNA comnprising the modified nucleotide sequence of SEQ IDNO: 602.Embodiment A63. A gRNA consisting of the modified nucleotide sequence of SEQ ID NO: 602.Embodiment A64. A lipid nanoparticle (LNP) composition comprising the gRNA of any one of embodiments A1-A63.Embodiment A65. A composition comprising the gRNA of any one of embodiments Al- A63 associated with a lipid nanoparticle (LNP).Embodiment A66. The composition of embodiment A64 or A65, wherein the LNP comprises a cationic lipid.Embodiment A67. The composition of embodiment A66, wherein the cationic lipid is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12- di enoate.Embodiment A68. The composition of any one of embodiments A64-A67, wherein the LNP comprises a helper lipid.Embodiment A69. The composition of embodiment A68, wherein the helper lipid is cholesterol.Embodiment A70. The composition of any one of embodiments A64-A69, wherein the LNP comprises a neutral lipid.Embodiment A71. The composition of embodiment A70, wherein the neutral lipid is 1,2- di stearoyl -sn-gly cero-3 -phosphocholine (D SPC) .Embodiment A72. The composition of any one of embodiments A64-A71, wherein the LNP comprises a stealth lipid.Embodiment A73. The composition of embodiment A72, wherein the stealth lipid is 1,2- dimyristoyl-rac-gly cero-3 -methoxypolyethylene glycol -2000 (PEG2k-DMG).Embodiment A74. The composition of any one of embodiments 64-73, wherein the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12- di enoate, DSPC, cholesterol, and PEG2k-DMG.Embodiment A75. A composition comprising the gRNA of any one of embodiments Al- A63, or the composition of any one of embodiments A64-A74, further comprising aCas nuclease or a nucleic acid which encodes a Cas nuclease, optionally a S. pyogenes Cas9 (SpyCas9) nuclease or a nucleic acid which encodes a SpyCas9 nuclease.Embodiment A76. The composition of embodiment A75, wherein the nuclease is a cleavase.Embodiment A77. The composition of embodiment A75, wherein the nuclease is a nickase or a dCas.Embodiment A78. The composition of embodiment A75, wherein the Cas nuclease comprises an amino acid sequence at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1089, 1092, or 1095.Embodiment A79. The composition of embodiment A75, wherein the Cas nuclease comprises the amino acid sequence of SEQ ID NO: 1092.Embodiment A80. The composition of any one of embodiments A75-A79, wherein the nucleic acid which encodes the Cas nuclease is an mRNA.Embodiment A81. The composition of embodiment A80, wherein the mRNA comprises a nucleotide sequence at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1090, 1091, 1093, or 1094.Embodiment A82. The composition of embodiment A80, wherein the mRNA comprises a nucleotide sequence at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1090.Embodiment A83. The composition of any one of embodiments A80-A82, wherein at least 10% of the uridine of the mRNA is substituted with a modified uridine.Embodiment A84. The composition of any one of embodiments A80-A83, wherein 10%- 25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90- 100% of the uridine of the mRNA is substituted with a modified uridine.Embodiment A85. The composition of any one of embodiments A80-A84, wherein at least 90% of the uridine of the mRNA is substituted with a modified uridine.Embodiment A86. The compposition of any one of embodiments A80-A85, wherein 100% of the uridine is substituted with a modified uridine.Embodiment A87. The composition of any one of embodiments A80-A86, wherein the modified uridine is one or more of Nl-methyl-pseudouridine, pseudouridine, 5- methoxyuridine, or 5-iodouridine.Embodiment A88. The composition of embodiment A87, wherein the modified uridine is Nl-methyl-pseudouridine.Embodiment A89. A system comprising the gRNA of any one of embodiments A1-A63 or the composition of any one of embodiments A64-A88.Embodiment A90. A kit comprising the gRNA of any one of embodiments A1-A63 or the composition of any one of embodiments A64-A88.Embodiment A91. A pharmaceutical formulation comprising the gRNA of any one of embodiments A1-A63 or the composition of any one of embodiments A64-A88, and a pharmaceutically acceptable carrier.Embodiment A92. A method of modifying a target DNA comprising, delivering any one or more of the following to a cell: i. the gRNA of any one of embodiments A1-A63 and a Cas nuclease or nucleic acid encoding a Cas nuclease, optionally a S. pyogenes Cas9 (SpyCas9) nuclease or a nucleic acid which encodes a SpyCas9 nuclease; ii. the composition of any one of embodiments A64-A88; iii. the system of embodiment A89; iv. the kit of embodiment A90; or v. the pharmaceutical formulation of embodiment A91.Embodiment A93. The gRNA of any one of embodiments A1-A63, the composition of any one of embodiments A64-A88, the system of embodiment A89, the kit of embodiment A90, or the pharmaceutical formulation of embodiment A91 for use in modifying a target DNA in a cell or for use in preparing a medicament for treating a disease or disorder.Embodiment A94.Use of the gRNA of any one of embodiments A1-A63, the composition of any one of embodiments A64-A88, the system of embodiment A89, the kit of embodiment A90, or the pharmaceutical formulation of embodiment A91 in the manufacture of a medicament for treating a disease or disorder.Embodiment A95. The gRNA of any one of embodiments A1-A63, the composition of any one of embodiments A64-A88, the system of embodiment A89, the kit of embodiment A90, or the pharmaceutical formulation of embodiment A91 for use in inducing a double-strand break within a PCSK9 gene in a cell or for reducing expression of a PCSK9 gene in a cell.Embodiment A96. The gRNA, composition, system, kit, or pharmaceutical composition of embodiment A95, wherein the cell is a liver cell, optionally a human liver cell.Embodiment A97. The gRNA, composition, system, kit, or pharmaceutical composition of embodiment A96, wherein the liver cell is a hepatocyte.Embodiment A98. The gRNA, composition, system, kit, or pharmaceutical composition of any one of embodiments A95-A97, wherein the cell is in a subject.Embodiment A99. The gRNA, composition, system, kit, or pharmaceutical composition of any one of embodiments A95-A98 for treating a subject having a PCSK9 related disease.Embodiment A100. A method of inducing a double-strand break within a PCSK9 gene in a cell or reducing expression of a PCSK9 protein in a cell comprising contacting a cell with the gRNA of any one of embodiments A1-A63, the composition of any one of embodiments A64-A88, the system of embodiment A89, the kit of embodiment A90, or the pharmaceutical formulation of embodiment A91.Embodiment A101. A method of modifying a genomic locus in a human liver cell, the method comprising contacting a human liver cell with the gRNA of any one of embodiments A1-A63, the composition of any one of embodiments A64-A88, the system of embodiment A89, the kit of embodiment A90, or the pharmaceutical formulation of embodiment A91.Embodiment A102. The method of embodiment A100 or A101, wherein the method is performed in vivo.Embodiment Al 03. A method of treating a PCSK9 related disease in a subject, the method comprising administering to the subject the gRNA of any one of embodiments A1-A63, the composition of any one of embodiments A64-A88, the system of embodiment A89, the kit of embodiment A90, or the pharmaceutical formulation of embodiment A91.Embodiment A104. The method of embodiment A103, further comprising determining the PCSK9 protein level in a subject blood or serum sample before and / or after the administering.Embodiment A105. A system for editing PCSK9 gene or for treating a PCSK9 related disease in a subject, comprising the gRNA of any one of embodiments Al- A63, the composition of any one of embodiments A64-A88, the system of embodiment A89, the kit of embodiment A90, or the pharmaceutical formulation of embodiment A91.

[0339] Further exemplary embodiments are included throughout the disclosure, including in the examples and claims that follow.Table 5. Table of sequencesIt is understood that if a DNA sequence (comprising Ts) is referenced with respect to an RNA, then Ts should be replaced with Us (which may be modified or unmodified depending on the context), and vice versa. In certain sequences, denotes that the nucleotide is linked to the next nucleotide with a phosphorothioate (PS) linkage, a lower case “m” denotes that the nucleotide is modified with a 2’-O-methyl (2’-0-Me) modfication, and a lower case “f ’ denotes that the nucleotide is modified with a 2’-fluoro (2’-F) modification. In certain sequences, an “N” or“n” denotes any nucleotide.EXAMPLES

[0340] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.Example 1. Materials and Methods1.1. In vitro transcription (“IVT”) of nuclease mRNA

[0341] Capped and polyadenylated mRNA containing N1 -methyl pseudo-U was generated by in vitro transcription using routine methods. Typically, a DNA plasmid containing a T7 promoter, a sequence for transcription, and a polyadenylation region was linearized with Xbal per manufacturer’s protocol. The Xbal was inactivated by heating. The linearized plasmid was purified from enzyme and buffer salts. The IVT reaction to generate modified mRNA was performed by incubating at 37°C: 50 ng / pL linearized plasmid; 2-5 mM each of GTP, ATP, CTP, and N1 -methyl pseudo-UTP (Trilink); 10-25 mM ARC A (Trilink);5 U / pL T7 RNA polymerase; 1 U / pL murine RNase inhibitor (NEB); 0.004 U / pL inorganic E. coli pyrophosphatase (NEB); and lx reaction buffer. TURBO DNase (Thermo Fisher) was added to a final concentration of O.OlU / pL, and the reaction was incubated at 37°C to remove the DNA template.

[0342] The mRNA was purified using a MegaClear Transcription Clean-up kit (Thermo Fisher) or a RNeasy Maxi kit (Qiagen) per the manufacturers' protocols. Alternatively, the mRNA was purified through a precipitation protocol, which in some cases was followed by HPLC-based purification. Briefly, after the DNase digestion, mRNA was purified using LiCl precipitation, ammonium acetate precipitation, and sodium acetate precipitation. For HPLC purified mRNA, after the LiCl precipitation and reconstitution, the mRNA was purified by RP-IP HPLC (see, e.g., Kariko, et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 el42). The fractions chosen for pooling were combined and desalted by sodium acetate / ethanol precipitation as described above. In a further alternative method, mRNA was purified with a LiCl precipitation method followed by further purification by tangential flow filtration. RNA concentrations were determined by measuring the light absorbance at 260 nm (Nanodrop), and transcripts were analyzed by capillary electrophoresis by Bioanalyzer (Agilent).

[0343] Streptococcus pyogenes (“Spy”) Cas9 mRNA was generated from plasmid DNA encoding an open reading frame according to any one of SEQ ID Nos: 1091 and 1094 (see sequences in Table 5). When the sequences cited in this paragraph are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which can bemodified nucleosides as described above). Messenger RNAs used in the Examples include a 5' cap and a 3' polyadenylation sequence, e.g., up to 100 nts. Guide RNAs were chemically synthesized by commercial vendors or using standard in vitro synthesis techniques with modified nucleotides.1.2. Preparation of LNP formulation containing sgRNA and Cas9 mRNA

[0344] In general, the lipid nanoparticle components were dissolved in 100% ethanol at various molar ratios. The RNA cargos (e.g., Cas9 mRNA and sgRNA) were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg / mL. The LNPs used contained ionizable lipid ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate), also called herein Lipid A, cholesterol, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and l,2-dimyristoyl-rac-glycero-3 -methoxypoly ethylene glycol-2000 (PEG2K-DMG) (e.g., catalog # GM-020 from NOF, Tokyo, Japan) in a molar ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a ratio of gRNA to mRNA of 1 :2 by weight. The LNPs used comprised a single RNA species such as Cas9 mRNA or an sgRNA. LNPs were similarly prepared with a mixture of Cas9 mRNA and a guide RNA.

[0345] The LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solution and one volume of water. First, the lipid in ethanol was mixed through a mixing cross with the two volumes of RNA solution. Then, a fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See W02016010840 FIG. 2). The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1 : 1 v / v). Diluted LNPs were buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) and concentrated as needed by methods known in the art. The resulting mixture was then filtered using a 0.2 pm sterile filter. The final LNPs were characterized to determine the encapsulation efficiency, poly dispersity index, and average particle size. The final LNP was stored at 4°C or -80°C until further use.1.3. Next-generation sequencing (“NGS”) and analysis for editing efficiency

[0346] To quantitatively determine the efficiency of editing at the target location in the genome, sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing. PCR primers were designed around the target site within the gene of interest (e.g., PCSK9), and the genomic area of interest was amplified. Primer sequence design was done as is standard in the field.

[0347] Additional PCR was performed according to the manufacturer's protocols (Illumina) to add chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the reference genome (e.g., hg38) after eliminating those having low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion or deletion (“indel”) was calculated.

[0348] The editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of sequence reads with insertions or deletions (“indels”) over the total number of sequence reads, including wild type reads.1.3.1 Indel Analysis

[0349] The number of wild-type reads versus the number of reads which contained indels was calculated. Insertions and deletions were scored in a ~20 bp region centered on the predicted Cas9 cleavage site. Indel percentage is defined as the total number of sequencing reads with one or more bases inserted or deleted within the ~20 bp scoring region divided by the total number of sequencing reads, including wild type.Example 2. In vitro editing in Primary Hepatocytes

[0350] Single-guide RNAs (sgRNAs) respectively targeting the human PCSK9 gene with the spacer sequence of SEQ ID NO: 1, as shown in Tables 4A and 5, were lipofected into primary human (PHH) hepatocytes. Lipofection of Cas9 mRNA and gRNAs used premixed lipid formulations. The lipofection reagent contained ionizable lipid ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate), also called herein Lipid A, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50% Lipid A,38% cholesterol, 9% DSPC, and 3% PEG2k-DMG. This mixture was reconstituted in 100% ethanol then mixed with RNA cargos e.g., Cas9 mRNA (SEQ ID NO: 1090) and gRNA) at a lipid amine to RNA phosphate (N:P) molar ratio of about 6 to produce lipid nucleic acid mixtures. An mRNA comprising a Cas9 ORF of Table 5 was produced by in vitro transcription (IVT) as described in W02019 / 067910, see e.g., 354, using a 2-hour IVT reaction time and purifying the mRNA by LiCl precipitation followed by tangential flow filtration.

[0351] PHH (Gibco, Lot #9396) cells were used and plated at densities of 40,000 and 33,000 cells / well, respectively. Lipofection samples were prepared using an N:P molar ratio of about 7 and a gRNA:mRNA ratio of 6.5: 1 by weight. Cells were incubated at 37°C, 5% CO2 for 24 hours prior to treatment with the lipid nucleic acid mixtures. Lipid nucleic acid mixtures were incubated in media containing 10% fetal bovine serum (FBS) at 37°C for 10 minutes. Post-incubation, the lipid nucleic acid mixtures comprising 50ng of Cas9 mRNA were added to the cells. The cells were lysed 72 hours post-treatment for NGS analysis as described in Example 1. Mean editing results with standard deviation (SD) are shown in Table 6 for PHH. Samples were run in duplicate.Table 6: In vitro editing in PHHExample 3. In vitro editing in primary human hepatocytes with dilution curve

[0352] Guide RNAs targeting PCSK9 synthesized with the same spacer sequence and different scaffold constant region modification patterns and sequences (as shown in Table 4A) were tested for editing efficacy in primary human hepatocytes (PHH) (Gibco / Thermo Fisher, Lot: HU8373A).Example 3.1. PHH Cell preparation

[0353] PHH were thawed and resuspended in hepatocyte thawing medium followed by centrifugation. Supernatant was discarded and the pellet was resuspended in hepatocyte plating medium (William’s E Medium (Gibco, Cat. A12176-01) containing dexamethasone + plating cocktail supplement (Gibco, Cat. A15563, Lot 2019842) and FBS content (Gibco, Cat. A13450)). Cells were counted and plated with a density of 33,000 cells / well on Bio-coat collagen I coated 96-well plates (Coming, Cat. 354407). Plated cells were allowed to settle and adhere for 4-6 hours in a tissue culture incubator at 37°C and 5% CO2 atmosphere. After incubation cells were checked for monolayer formation and were washed once with hepatocyte maintenance medium (William’s E Medium (Gibco, Cat. A12176-01) containing dexamethasone + maintenance cocktail supplement (Gibco, Cat. Al 5564)) and incubated overnight.Example 3.2. LNP Treatment and Editing

[0354] LNPs were generally prepared as described in Example 1. The LNPs contained 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG by molar ratio. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a ratio of gRNA to mRNA (SEQ ID NO: 1090) of 1 :2 by weight. Each LNP was incubated in William's E Medium (Gibco, A1217601) with maintenance supplements and 3%fetal bovine serum that was applied to cells using a 12-point dose response assay starting with a LNP dose of 450 ng total RNA by weight.

[0355] Samples were run in triplicate. After 72 hours, cells were harvested and analyzed by NGS as described in Example 1. Dose response curves are plotted in FIG. 1. EC50 values and mean percent editing results are shown in Table 7.Table 7: Editing efficiency and EC50 (nM) for selected guides

[0356] For guideRNAs having the % editing at top dose that was significantly lower (>5% difference) than the % editing at the dose resulting in max editing, the top dose editing value was excluded from EC50 calculations.Table 8. Maximum editing efficiency for selected guidesExample 4. In vivo editing using different guide formats in a humanized PCSK9 mouse model using lipid nanoparticles (LNPs)

[0357] Selected guide RNAs from Table 8 were tested for editing efficiency in vivo.Male transgenic mice comprising a h...

Claims

We claim:

1. A guide RNA (gRNA) comprising, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’: a. a lower stem region portion being nucleotides LSI to LS6, b. a bulge region portion being nucleotides B 1 to B2, c. an upper stem region being nucleotides US1 to US 12, optionally wherein the upper stem region further comprises up to 8 additional nucleotides, d. a bulge region portion being nucleotides B3 to B6, e. a lower stem region portion being nucleotides LS7 to LS12, f. a nexus region being nucleotides N1 to N18, and g. a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ : iv. a hairpin 1 region being nucleotides Hl-1 to Hl-12, optionally wherein the hairpin 1 region lacks up to 6 nucleotides of Hl-1 to Hl-12, v. a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and vi. a hairpin 2 region being nucleotides H2-1 to H2-15; and h. optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: i. a 5’ end modification, j. modified nucleotides at 10, 11, or all nucleotides in the upper stem region, k. 3 or more modified nucleotides in the hairpin 2 region, wherein the gRNA further comprises one or more additional modifications selected from: l. modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; m. modified nucleotides at B2 and B3 of the bulge region; n. modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; o. modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11 of the hairpin 1 region, wherein the hairpin 1 region consists of nucleotides Hl-2, Hl -5, Hl-6, Hl-7, Hl-8 and Hl-11; p. modified nucleotides at all nucleotides of the hairpin 1 region, wherein the hairpin 1 region comprises Hl-1 to Hl-12; andq. modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.

2. The gRNA of claim 1, wherein the conserved portion comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 400, SEQ ID NO: 709 or SEQ ID NO: 710.

3. The gRNA of claim 2, wherein the conserved portion comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 701, or 702.

4. The gRNA of claim 3, wherein the gRNA comprises a nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 801, 802, 809, or 810.

5. The gRNA of any one of claims 1-4, wherein the conserved portion comprises at least one region with unmodified and modified nucleotides selected from: a. the lower stem region comprising modified nucleotides at LSI, LS8, LS10, and LS12; nucleotides not modified with 2’-0-Me at LS2, LS3, LS4, LS5, optionally further comprising nucleotides not modified with 2’-0-Me at LS6 and / or LS9; b. the bulge region comprising nucleotides not modified with 2’-0-Me at B5 and B6; c. the upper stem region comprising modified nucleotides at at least 10 positions selected from US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, optionally at all positions US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; d. the nexus region comprising modified nucleotides at N1 , N2, N4, N7, Ni l, N12, and N17; and nucleotides not modified with 2’-O-Me at N6, N8, N9, N10, N13, and N14, optionally further comprising a nucleotide not modified with 2’-O-Me at N16; e. the hairpin 1 (Hl) region, comprising modified nucleotides at Hl-2, Hl-6, Hl- 7, Hl-8, and Hl-11; and nucleotides not modified with 2’-O-Me at Hl-5; f. the hairpin 2 (H2) region comprising modified nucleotides at H2-1, H2-2, H2- 3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15, optionally further comprising a modified nucleotide at H2-11; and nucleotides not modified with 2’-O-Me at H2-8, H2-9, and H2-10; andg. the n is a modified or unmodified nucleotide.

6. The gRNA of any one of claims 1-5, wherein the lower stem region comprises modified nucleotides at LSI, LS8, LS10, and LS12; and nucleotides not modified with 2’-0-Me at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1.

7. The gRNA of any one of claims 1-6, wherein the lower stem region comprises unmodified nucleotides at LS2, LS3, LS4, and LS5, optionally at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1.

8. The gRNA of any one of claims 1-7, wherein the lower stem region comprises unmodified nucleotides at LS7, LS9, and LSI 1.

9. The gRNA of any one of claims 1-8, wherein the bulge region comprises modified nucleotides at B2 and B3.

10. The gRNA of any one of claims 1-9, wherein the bulge region comprises unmodified nucleotides at B5 and B6, optionally at Bl, B4, B5, and B6.

11. The gRNA of any one of claims 1-10, wherein the nexus region comprises modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17.

12. The gRNA of any one of claims 1-11, wherein the nexus region comprises unmodified nucleotides atN6, N8, N9, N10, N13, N14, and N18, optionally atN3, N5, N6, N8, N9, N10, N13, N14, N15, and N18.

13. The gRNA of any one of claims 1-12, wherein the hairpin 1 region comprises modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11.

14. The gRNA of any one of claims 1-13, wherein the hairpin 1 region comprises an unmodified nucleotide at Hl -5.

15. The gRNA of any one of claims 1-14, wherein the hairpin 2 region comprises modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-25.

16. The gRNA of any one of claims 1-15, wherein the hairpin 2 region comprises unmodified nucleotides at H2-8, H2-9, and H2-10.

17. A guide RNA (gRNA) comprising, from 5’ to 3’, a spacer region of 17-20 nucleotides in length and a conserved portion, wherein the conserved portion comprises, from 5’ to 3’: a. a lower stem region portion being nucleotides LSI to LS6, b. a bulge region portion being nucleotides B 1 to B2, c. an upper stem region being nucleotides US 1 to US 12,d. a bulge region portion being nucleotides B3 to B6, e. a lower stem region portion being nucleotides LS7 to LS12, f. a nexus region being nucleotides N1 to N18, and g. a hairpin region, wherein the hairpin region comprises, from 5’ to 3’ :(i) a hairpin 1 region being nucleotides Hl -2, Hl -5, Hl -6, Hl -7, Hl-8, and Hl-11,(ii) a single nucleotide between hairpin 1 region and hairpin 2 region being nucleotide n, and(iii) a hairpin 2 region being nucleotides H2-1 to H2-15, and h. optionally, a 3’ tail region being one or more nucleotides, wherein the gRNA further comprises: i. a 5’ end modification, j . modified nucleotides at all nucleotides in the upper stem region, k. modified nucleotides at B2 and B3 of the bulge region; l. modified nucleotides at LSI, LS8, LS10, and LS12 of the lower stem region; m. modified nucleotides at Nl, N2, N4, N7, Ni l, N12, and N17 of the nexus region; n. modified nucleotides at Hl-2, Hl-6, Hl-7, Hl-8, and Hl-11 of the hairpin 1 region; and o. modified nucleotides at H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-12, H2-13, H2-14, and H2-15 of the hairpin 2 region.

18. The gRNA of claim 17, wherein: a. the lower stem region comprises unmodified nucleotides at LS2, LS3, LS4, and LS5, optionally at LS2, LS3, LS4, LS5, LS6, LS7, LS9, and LSI 1; b. the bulge region comprises unmodified nucleotides at B5 and B6, optionally at B1, B4, B5, and B6; c. the nexus region comprises unmodified nucleotides at N6, N8, N9, N10, N13, N14, and N18, optionally at N3, N5, N6, N8, N9, N10, N13, N14, N15, and N18; d. the hairpin 1 region comprises an unmodified nucleotide at Hl-5; and e. the hairpin 2 region comprises unmodified nucleotides at H2-8, H2-9, and H2- 10.

19. The gRNA of any one of claims 1-18, wherein the spacer sequence comprises one or more modified nucleotide.

20. The gRNA of any one of claims 1-19, wherein the spacer sequence comprises one or more phosphorothioate (PS) modifications.

21. The gRNA of any one of claims 1-20, wherein the spacer sequence comprises one or more modified nucleotide selected from a 2’-O-methyl (2’-O-Me) modified nucleotide or a 2’-fluoro (2’-F) modified nucleotide.

22. The gRNA of any one of claims 1-21, wherein the spacer sequence is 20 nucleotides in length.

23. The gRNA of any one of claims 1-22, wherein the spacer sequence comprises 2’-O- Me modified nucleotides at each of the first three 5’ terminal nucleotides (SI, S2, and S3).

24. The gRNA of any one of claims 1-23, wherein the spacer sequence comprises one or more phosphorothioate (PS) linkages between the first four 5’ terminal nucleotides.

25. The gRNA of any one of claims 22-24, wherein the spacer sequence comprises modified nucleotides at SI, S2, and S3; and nucleotides not modified with 2’-0-Me at S5, S6, S7, S12, S15, S16, and S19; optionally comprising nucleotides not modified with 2’-0-Me at S5, S6, S7, S8, S12, S15, S16, and S19.

26. The gRNA of any one of claims 23-25, wherein the spacer sequence further comprises modified nucleotides at S9 and S13.

27. The gRNA of claim 26, wherein the spacer sequence comprises modified nucleotides at SI, S2, S3, S4, S7, S8, S9, S10, SI 1, S13, S14, S17 and S18.

28. The gRNA of any one of claims 1-27, wherein at least one modified nucleotide comprises a modified nucleotide that stabilizes A-form helix structure, optionally wherein each modified nucleotide comprising a 2’-ribose modification comprises a modified nucleotide that stabilizes A-form helix structure.

29. The gRNA of any one of claims 1-28, wherein each modified nucleotide comprises a modification independently selected from a 2’-O-methyl (2’-0-Me) modified nucleotide, a 2’ -O-(2 -methoxy ethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’- F) modified nucleotide, a bicyclic ribose analog, a locked nucleic acid (LNA), ethylene-bridged nucleic acids (ENA), a bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), and a phosphorothioate (PS) modification.

30. The gRNA of any one of claims 1-29, wherein at least one modified nucleotide comprising a 2’ -ribose modification further comprises a phosphorothioate modified nucleotide, optionally a 2’-0-Me modified nucleotide and a phophosphorothioate modified nucleotide.

31. The gRNA of any one of claims 1-30, wherein each nucleotide not modified with 2’- O-Me is independently selected from a 2’-fluoro (2’-F) modified nucleotide and an unmodified nucleotide.

32. The gRNA of any one of claims 1-31, wherein the modified nucleotides of the upper stem region are 2’-0-Me modified nucleotides.

33. The gRNA of any one of claims 1-32, wherein the modified nucleotides of the lower stem region, bulge, nexus region, hairpin 1 region, or hairpin 2 region, and optionally each of the lower stem region, bulge, nexus region, hairpin 1 region, and hairpin 2 region are 2’-0-Me modified nucleotides.

34. The gRNA of any one of claims 1-33, wherein the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 102, 107, 103, 104, 108, 109, 110, 111, 112, 122, 127, 123, 124, 128, 129, 130, 131, and 132.

35. The gRNA of any one of claims 1-33, wherein the conserved portion comprises a modified nucleotide sequence of any one of SEQ ID NOs: 202, 207, 203, 204, 208, 209, 210, 211, 212, 222, 227, 223, 224 228, 229, 230, 231, and 232.

36. The gRNA of any one of claims 1-33, wherein the conserved portion comprises a modified nucleotide sequence of SEQ ID NO: 102, 202, 122, or 222.

37. The gRNA of any one of claims 1-36, wherein the gRNA comprises a 3’ end modification.

38. The gRNA of claim 37, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are modified nucleotides.

39. The gRNA of claim 37 or 38, wherein the last three nucleotides at the 3’ end of the gRNA are modified nucleotides.

40. The gRNA of any one of claims 37-39, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are 2’-0-Me modified nucleotides.

41. The gRNA of any one of claims 37-40, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are linked with phosphorothioate (PS) bonds.

42. The gRNA of any one of claims 37-41, wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are 2’-0-Me modified nucleotides, further wherein the last two, three, or four nucleotides at the 3’ end of the gRNA are linked with phosphorothioate (PS) bonds.

43. The gRNA of any one of claims 1-42, further comprising a 3’ tail, optionally wherein the 3’ tail is 1, 2, 3, or 4 nucleotides, optionally 1 nucleotide in length.

44. The gRNA of claim 43, wherein the 3’ tail comprises a modified nucleotide at any one or more of the nucleotides present in the 3’ tail, optionally wherein the modified nucleotide is a 2’-0-Me modified nucleotide or a phosphorothioate modified nucleotide.

45. The gRNA of claim 44, wherein the 3’ tail is fully modified.

46. The gRNA of any one of claims 43-45, wherein the 3’ tail comprises a terminal 2’-O- Me modified uridine nucleotide wherein the terminal 2’-0-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage.

47. The gRNA of any one of claims 1-2 or 5-42, wherein the gRNA does not comprise a 3’ tail.

48. The gRNA of any one of claims 1-47, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are modified nucleotides.

49. The gRNA of claim 48, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are 2’-0-Me modified nucleotides.

50. The gRNA of claim 48 or 49, wherein the first three nucleotides at the 5’ end of the gRNA are modified nucleotides.

51. The gRNA of any one of claims 48-50, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are linked with phosphorothioate (PS) bonds.

52. The gRNA of any one of claims 48-51, wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are 2’-0-Me modified nucleotides, further wherein the first two, three, or four nucleotides at the 5’ end of the gRNA are linked with phosphorothioate (PS) bonds.

53. A gRNA comprising a modified nucoleotide sequence selected from: a. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 302, 402, 322, and 422; b. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 307, 407, 327, and 427; c. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 303, 403, 304, 404, 323, 423, 324, and 424; and d. a nucleotide sequence at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 308, 408, 309,409, 310, 410, 311, 411, 312, 412, 328, 428, 329, 429, 330, 430, 331, 431, 332, and 432.

54. A gRNA comprising a modified nucleotide sequence that is at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 302, 402, 322, and 422.

55. A gRNA comprising a modified nucleotide sequence selected from SEQ ID Nos: 302 and 402, and further comprising a 3’ tail, optionally wherein the 3’ tail is 1, 2, 3, or 4, nucleotides, and optionally wherein the 3’ tail comprises a terminal 2’-O-Me modified uridine nucleotide wherein the terminal 2’-O-Me modified uridine nucleotide is linked to a penultimate nucleotide by a phosphorothioate linkage.

56. A gRNA comprising a modified nucleotide sequence selected from SEQ ID NOs: 322 and 422.

57. The gRNA of any one of claims 1-56, further comprising a spacer comprising the nucleotide sequence of SEQ ID NO: 1.

58. A gRNA consisting of the modified nucleotide sequence of SEQ ID NOs: 322 or 422.

59. A gRNA consisting of the modified nucleotide sequence of SEQ ID NO: 322 or 422, wherein the nucleotide sequence of the first 20 nucleotides is the sequence of SEQ ID NO: 1.

60. A gRNA comprising the modified nucleotide sequence of any one of SEQ ID NOs: 601-619.

61. A gRNA consisting of the modified nucleotide sequence of any one of SEQ ID NOs: 601-619.

62. A gRNA comnprising the modified nucleotide sequence of SEQ ID NO: 602.

63. A gRNA consisting of the modified nucleotide sequence of SEQ ID NO: 602.

64. A lipid nanoparticle (LNP) composition comprising the gRNA of any one of claims 1- 63.

65. A composition comprising the gRNA of any one of claims 1-63 associated with a lipid nanoparticle (LNP).

66. The composition of claim 64 or 65, wherein the LNP comprises a cationic lipid.

67. The composition of claim 66, wherein the cationic lipid is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12- di enoate.

68. The composition of any one of claims 64-67, wherein the LNP comprises a helper lipid.

69. The composition of claim 68, wherein the helper lipid is cholesterol.

70. The composition of any one of claims 64-69, wherein the LNP comprises a neutral lipid.

71. The composition of claim 70, wherein the neutral lipid is l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).

72. The composition of any one of claims 64-71, wherein the LNP comprises a stealth lipid.

73. The composition of claim 72, wherein the stealth lipid is 1,2-dimyristoyl-rac-glycero- 3 -methoxypolyethylene gly col-2000 (PEG2k-DMG).

74. The composition of any one of claims 64-73, wherein the LNP comprises (9Z,12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12- di enoate, DSPC, cholesterol, and PEG2k-DMG.

75. A composition comprising the gRNA of any one of claims 1-63, or the composition of any one of claims 64-74, further comprising a Cas nuclease or a nucleic acid which encodes a Cas nuclease, optionally a S. pyogenes Cas9 (SpyCas9) nuclease or a nucleic acid which encodes a SpyCas9 nuclease.

76. The composition of claim 75, wherein the nuclease is a cleavase.

77. The composition of claim 75, wherein the nuclease is a nickase or a dCas.

78. The composition of claim 75, wherein the Cas nuclease comprises an amino acid sequence at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1089, 1092, or 1095.

79. The composition of claim 75, wherein the Cas nuclease comprises the amino acid sequence of SEQ ID NO: 1092.

80. The composition of any one of claims 75-79, wherein the nucleic acid which encodes the Cas nuclease is an mRNA.

81. The composition of claim 80, wherein the mRNA comprises a nucleotide sequence at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1090, 1091, 1093, or 1094.

82. The composition of claim 80, wherein the mRNA comprises a nucleotide sequence at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1090.

83. The composition of any one of claims 80-82, wherein at least 10% of the uridine of the mRNA is substituted with a modified uridine.

84. The composition of any one of claims 80-83, wherein 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine of the mRNA is substituted with a modified uridine.

85. The composition of any one of claims 80-84, wherein at least 90% of the uridine of the mRNA is substituted with a modified uridine.

86. The compposition of any one of claims 80-85, wherein 100% of the uridine is substituted with a modified uridine.

87. The composition of any one of claims 80-86, wherein the modified uridine is one or more of Nl-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.

88. The composition of claim 87, wherein the modified uridine is Nl-methyl- pseudouridine.

89. A system comprising the gRNA of any one of claims 1-63 or the composition of any one of claims 64-88.

90. A kit comprising the gRNA of any one of claims 1-63 or the composition of any one of claims 64-88.

91. A pharmaceutical formulation comprising the gRNA of any one of claims 1-63 or the composition of any one of claims 64-88, and a pharmaceutically acceptable carrier.

92. A method of modifying a target DNA comprising, delivering any one or more of the following to a cell: i. the gRNA of any one of claims 1-63 and a Cas nuclease or nucleic acid encoding a Cas nuclease, optionally a S. pyogenes Cas9 (SpyCas9) nuclease or a nucleic acid which encodes a SpyCas9 nuclease; ii. the composition of any one of claims 64-88; iii. the system of claim 89; iv. the kit of claim 90; or v. the pharmaceutical formulation of claim 91.

93. The gRNA of any one of claims 1-63, the composition of any one of claims 64-88, the system of claim 89, the kit of claim 90, or the pharmaceutical formulation of claim 91 for use in modifying a target DNA in a cell or for use in preparing a medicament for treating a disease or disorder.

94. Use of the gRNA of any one of claims 1-63, the composition of any one of claims 64- 88, the system of claim 89, the kit of claim 90, or the pharmaceutical formulation of claim 91 in the manufacture of a medicament for treating a disease or disorder.

95. The gRNA of any one of claims 1-63, the composition of any one of claims 64-88, the system of claim 89, the kit of claim 90, or the pharmaceutical formulation of claim 91 for use in inducing a double-strand break within a PCSK9 gene in a cell or for reducing expression of a PCSK9 gene in a cell.

96. The gRNA, composition, system, kit, or pharmaceutical composition of claim 95, wherein the cell is a liver cell, optionally a human liver cell.

97. The gRNA, composition, system, kit, or pharmaceutical composition of claim 96, wherein the liver cell is a hepatocyte.

98. The gRNA, composition, system, kit, or pharmaceutical composition of any one of claims 95-97, wherein the cell is in a subject.

99. The gRNA, composition, system, kit, or pharmaceutical composition of any one of claims 95-98 for treating a subject having a PCSK9 related disease.

100. A method of inducing a double-strand break within a PCSK9 gene in a cell or reducing expression of a PCSK9 protein in a cell comprising contacting a cell with the gRNA of any one of claims 1-63, the composition of any one of claims 64-88, the system of claim 89, the kit of claim 90, or the pharmaceutical formulation of claim 91.

101. A method of modifying a genomic locus in a human liver cell, the method comprising contacting a human liver cell with the gRNA of any one of claims 1-63, the composition of any one of claims 64-88, the system of claim 89, the kit of claim 90, or the pharmaceutical formulation of claim 91.

102. The method of claim 100 or 101, wherein the method is performed in vivo.

103. A method of treating a PCSK9 related disease in a subject, the method comprising administering to the subject the gRNA of any one of claims 1-63, the composition of any one of claims 64-88, the system of claim 89, the kit of claim 90, or the pharmaceutical formulation of claim 91.

104. The method of claim 103, further comprising determining the PCSK9 protein level in a subject blood or serum sample before and / or after the administering.

05. A system for editing PCSK9 gene or for treating a PCSK9 related disease in a subject, comprising the gRNA of any one of claims 1-63, the composition of any one of claims 64-88, the system of claim 89, the kit of claim 90, or the pharmaceutical formulation of claim 91.

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