Modified guide RNA

Abstract

The present invention relates to modified single guide RNA and double guide RNA having improved in vitro and in vivo activity in a gene editing method.

Description

[0001] This application is a divisional application of Chinese patent application 201780085888.5 (PCT / US2017 / 065306), whose application date is December 8, 2017 and whose invention name is “Modified Guide RNA”.

[0002] This application contains a sequence listing, which has been submitted electronically in ASCII format, and its entire contents are incorporated herein by reference. This ASCII copy, created on December 7, 2017, is named 01155-0004-00PCT_SeqList.txt and is 118,877 bytes in size.

[0003] This application claims priority to U.S. Provisional Application No. 62 / 431,756, filed on December 8, 2016, which is incorporated by reference in its entirety.

[0004] The present invention relates to the field of gene editing using the CRISPR / Cas system, which is part of the prokaryotic immune system used to recognize and cut exogenous genetic elements. The CRISPR / Cas system relies on a single nuclease called CRISPR-associated protein 9 (Cas9), which induces site-specific breaks in DNA. Cas9 is guided to a specific DNA sequence by a small RNA molecule called guide RNA (gRNA). Guide RNA includes trRNA (also called tracrRNA) and crisprRNA (crRNA). trRNA and crRNA can be contained in a single guide RNA (sgRNA) or in two independent RNA molecules of a dual guide RNA (dgRNA). Cas9 and the combination with trRNA and crRNA or sgRNA is called Cas9 ribonucleoprotein complex (RNP).

[0005] Oligonucleotides, especially RNA, are sometimes cleaved by endonucleases or exonucleases and degraded in cells and serum. Improved methods and compositions are needed to prevent such degradation, improve gRNA stability, and enhance gene editing efficiency, especially for therapeutic applications. SUMMARY OF THE INVENTION

[0007] In some embodiments, a therapeutic genome editing tool comprising a modified guide RNA is provided. The modified guide RNA described herein can improve the stability of the guide RNA and the guide RNA / Cas9 complex, and improve the activity of Cas9 (e.g., SpyCas9 and equivalents) in cleaving the target DNA. In some embodiments, the guide RNA is sgRNA. In some embodiments, the guide RNA is dgRNA. In some embodiments, the guide RNA is tracrRNA. In some embodiments, the guide RNA is crRNA.

[0008] The guide RNA described herein comprises at least one modified nucleotide. The modification may include 2'-O-methyl (2'-O-Me), '-O-(2-methoxyethyl) (2'-O-moe), 2'-fluoro (2'-F), phosphorothioate (PS) bonds between nucleotides, GC substitutions, and reverse abasic linkages between nucleotides and their equivalents. Embodiments of the present invention include:

[0009] In some embodiments, a single guide RNA (sgRNA) is contemplated that comprises a 5' end modification and one or more modifications of one or more of the following: an upper stem region; a hairpin 1 region; and a hairpin 2 region, wherein the 5' end modification comprises at least two phosphorothioate bonds within the first seven nucleotides at the 5' end of the 5' terminus. In some cases, the modification is a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the modification is a 2'-fluoro (2'-F) modified nucleotide.

[0010] In some embodiments, the sgRNA comprises modifications at US1 to US12 and / or modifications at H1-1 and / or modifications in H2-1. In some embodiments, the sgRNA comprises modifications at H1-1 to H1-12 and / or H2-1 to H2-15. In some embodiments, the sgRNA comprises one or more modifications at each of the upper stem region, the hairpin 1 region, and the hairpin 2 region. In some embodiments, the sgRNA comprises modified nucleotides between the hairpin 1 region and the hairpin 2 region. In some embodiments, the sgRNA comprises modifications in the lower stem region.

[0011] In some embodiments, the sgRNA comprises a modification at the 5' end and / or the 3' end. In some embodiments, the sgRNA comprises a 3' end modification in the 3' end. In some embodiments, the sgRNA comprises a modification at least two of the last four nucleotides at the 3' end of the 3' end. In some embodiments, the sgRNA comprises a 5' end modification in the 5' end. In some embodiments, the sgRNA comprises a modification of at least two of the first four nucleotides at the 5' end of the 5' end. In some embodiments, the sgRNA comprises a 3' end modification in the 3' end and a 5' end modification in the 5' end. In some embodiments, the sgRNA comprises a modification of at least two of the last four nucleotides at the 3' end of the 3' end and at least two of the first four nucleotides at the 5' end of the 5' end. In some cases, these modifications are 2'-O-Me, 2'-F, 2'-O-moe, or a phosphorothioate (PS) bond connecting nucleotides. In some embodiments, the sgRNA comprises a PS bond between at least two of the last four nucleotides at the 3' end of the 3' end and / or at least two of the first four nucleotides at the 5' end of the 5' end. In some cases, the sgRNA comprises a 5' end and a 3' end having more than one modification as described herein, such as a PS bond and a 2'-O-Me modification.

[0012] In some embodiments, the sgRNA comprises a modification in the bulge region. In some embodiments, 50% of the nucleotides in the bulge region are modified, wherein the modification is 2'-O-Me or 2'-F.

[0013] In some embodiments, the sgRNA comprises a modification in the nexus region. In some embodiments, the sgRNA comprises a modification at N15, N16, N17, and / or N18 in the nexus region, wherein the modification is 2'-O-Me or 2'-F. In some cases, N16, N17, and N18 are linked to a PS bond.

[0014] In some embodiments, the sgRNA comprises at least the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus that are modified.

[0015] In some embodiments, the sgRNA comprises a modification at the 3' end and / or the 5' end. In some cases, the first four nucleotides at the 5' end of the 5' end and the last four nucleotides at the 3' end of the 3' end are connected to a phosphorothioate (PS) bond. In some embodiments, the 5' and 3' modifications comprise 2'-O-Me or 2'-O-moe. In some embodiments, the 5' and 3' modifications comprise 2'-F. In some embodiments, the 5' and / or 3' modifications comprise a PS bond connecting nucleotides. In some embodiments, the 5' and / or 3' modifications comprise one or more of 2'-O-Me, 2'-O-moe, 2'-F, and a PS bond connecting nucleotides.

[0016] In some embodiments, the sgRNA comprises modifications at the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus. In some cases, these modifications are PS bonds connecting the first four and last four nucleotides. In some embodiments, the sgRNA further comprises 2'-O-Me modifications at the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus.

[0017] In some embodiments, the sgRNA comprises a modification at the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus, wherein the modification is at least a PS bond connecting the four nucleotides, and further wherein the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus comprise a 2'-O-Me, 2'-O-moe, or 2'-F modification.

[0018] In some embodiments, the sgRNA comprises modifications LS1, LS6, LS7, LS8, LS11, and LS12, wherein the modifications are 2'-O-Me or 2'-F.

[0019] In some embodiments, the sgRNA comprises a modification at each of the nucleotides in the bulge region, wherein the modification is 2'-O-Me or 2'-F.

[0020] In some embodiments, the sgRNA comprises a modification at each of the nucleotides in the upper stem region, wherein the modification is 2'-O-Me or 2'-F.

[0021] In some embodiments, the sgRNA comprises a modification at each of the nucleotides in the hairpin 1 region, wherein the modification is 2'-O-Me or 2'-F.

[0022] In some embodiments, the sgRNA comprises a modification at each of the nucleotides in the hairpin 2 region, wherein the modification is 2'-O-Me or 2'-F.

[0023] In some embodiments, sgRNAs are contemplated that comprise 2'-O-Me modified nucleotides at the following positions:

[0024] a. The first three nucleotides at the 5' end of the 5' terminus;

[0025] b. LS1, LS6, LS7, LS8, LS11 and / or LS12 in the lower stem region;

[0026] c. B1 and / or B2 in the protrusion area;

[0027] d. Each nucleotide in the upper stem region;

[0028] e. N16, N17 and / or N18 in the connecting region;

[0029] f. Each nucleotide in the hairpin 1 region;

[0030] g. each nucleotide in region 2 of the hairpin; and

[0031] h. The last four nucleotides at the 3' end of the 3' terminus.

[0032] In some embodiments, B3 to B6 are modified with 2'-O-Me. In some cases, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end, and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end. In some embodiments, the sgRNA comprises 2'-F modifications at LS9 and LS10. In some embodiments, the sgRNA comprises 2'F modifications at N15, N16, N17, and N18. In some embodiments, the sgRNA comprises 2'F modifications at H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15. In some embodiments, the sgRNA comprises 2'F modifications at the second to last, third to last, and fourth to last nucleotides at the 3' end of the 3' end.

[0033] In some embodiments, sgRNAs are contemplated that comprise 2'-F modified nucleotides at the following positions:

[0034] a. LS9 and LS10 in the lower stem region;

[0035] b. N15, N16, N17 and N18 in the connection area: and

[0036] c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14 and H2-15 in the hairpin 2 region.

[0037] In some embodiments, the sgRNA comprises 2'-F modified nucleotides at the second to last, third to last, and fourth to last nucleotides at the 3' terminus. In some embodiments, the sgRNA comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' terminus of the 5' terminus, and three PS bonds connecting the last four nucleotides at the 3' terminus of the 3' terminus. In some embodiments, the sgRNA comprises 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' terminus of the 5' terminus and 2'-O-Me or 2'-F modified nucleotides at three of the last four nucleotides at the 3' terminus of the 3' terminus.

[0038] In some embodiments, sgRNAs are contemplated that include:

[0039] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0040] b. Optional 2'-O-Me modified nucleotides at LS1 and / or LS6;

[0041] c. 2'-O-Me modified nucleotides at US to US12;

[0042] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0043] e. an optional 2'-O-Me modified nucleotide between hairpin 1 region and hairpin 2 region;

[0044] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0045] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus; and optionally

[0046] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0047] In some embodiments, sgRNAs are contemplated that include:

[0048] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0049] b. 2'-F modified nucleotides at LS1 to LS6;

[0050] c. 2'-O-Me modified nucleotides at US1 to US12;

[0051] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0052] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0053] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0054] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus; and optionally

[0055] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0056] In some embodiments, sgRNAs are contemplated that include:

[0057] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0058] b. 2'-F modified nucleotides at LS2 to LS5;

[0059] c. 2'-O-Me modified nucleotides at LS1 and LS6;

[0060] d. 2'-O-Me modified nucleotides at US1 to US12;

[0061] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0062] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0063] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0064] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end, and optionally

[0065] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0066] In some embodiments, sgRNAs are contemplated that include:

[0067] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0068] b. 2'-O-Me modified nucleotides at US1 to US12;

[0069] c. 2'-O-Me modified nucleotides at LS7, LS8, LS11 and LS12;

[0070] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0071] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0072] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0073] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end,

[0074] And optionally further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5' end of the 5' end and three PS bonds linking the last four nucleotides at the 3' end of the 3' end.

[0075] In some embodiments, sgRNAs are contemplated that include:

[0076] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0077] b. 2'-O-Me modified nucleotides at US1 to US12;

[0078] c. 2'-O-Me modified nucleotides at LS7, LS8, LS11 and LS12;

[0079] d. 2'-F modified nucleotides at LS9 and LS10;

[0080] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0081] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0082] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0083] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end,

[0084] And optionally further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5' end of the 5' end and three PS bonds linking the last four nucleotides at the 3' end of the 3' end.

[0085] In some embodiments, sgRNAs are contemplated that include:

[0086] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0087] b. 2'-O-Me modified nucleotides at US1 to US12;

[0088] c. 2'-O-Me modified nucleotides at LS8, LS10 and LS12;

[0089] d. 2'-OF modified nucleotides at LS7, LS9 and LS11;

[0090] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0091] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0092] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0093] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end, and optionally

[0094] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0095] In some embodiments, sgRNAs are contemplated that include:

[0096] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0097] b. 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11 and LS12

[0098] c. 2'-O-Me modified nucleotides at US1 to US12;

[0099] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0100] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0101] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0102] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end, and optionally

[0103] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0104] In some embodiments, sgRNAs are contemplated that include:

[0105] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0106] b. 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11 and LS12;

[0107] c. 2'-F modified nucleotides at LS9 and LS10;

[0108] d. 2'-O-Me modified nucleotides at US1 to US12;

[0109] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0110] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0111] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0112] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end, and optionally

[0113] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0114] In some embodiments, sgRNAs are contemplated that include:

[0115] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0116] b. 2'-O-Me modified nucleotides at US1 to US12;

[0117] c. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0118] d. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0119] e. 2'-O-Me modified nucleotides at H2-1 to H2-8;

[0120] f. 2'-F modified nucleotides at H2-9 to H2-15;

[0121] g. 2'-F modified nucleotides at the second to last, third to last and fourth to last nucleotides at the 3' terminus; and

[0122] h. A 2'-O-Me modified nucleotide at the last nucleotide at the 3' end, and optionally

[0123] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0124] In some embodiments, sgRNAs are contemplated that include:

[0125] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0126] b. 2'-O-Me modified nucleotides at US1 to US12;

[0127] c. 2'-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12;

[0128] d. 2'-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9 and H1-11;

[0129] e. 2'-F modified nucleotides between hairpin 1 and hairpin 2;

[0130] f. 2'-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14;

[0131] g. 2'-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13 and H2-15;

[0132] h. 2'-F modified nucleotides at the second to last and fourth to last nucleotides at the 3' end; and

[0133] i. 2'-O-Me modified nucleotides at the third to last and last nucleotides at the 3' end of the 3' terminus,

[0134] And optionally further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5' end of the 5' end and three PS bonds linking the last four nucleotides at the 3' end of the 3' end.

[0135] In some embodiments, sgRNAs are contemplated that include:

[0136] a. 2'-O-Me modified nucleotides at LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15; and

[0137] b. LS7, LS9, LS11; 2'-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12 and H2-14, and optionally

[0138] Further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end, and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end; and optionally further comprising:

[0139] c. 2'-O-Me modified nucleotides at the last and third to last nucleotides at the 3' end of the 3' terminus; and / or

[0140] d. 2'-F modified nucleotides at the second to last, fourth to last and / or last nucleotide at the 3' end of the 3' terminus.

[0141] In some embodiments, sgRNAs are contemplated that comprise a nucleic acid of any one of SEQ ID Nos: 228 to 353, including modifications of Table 4. In some embodiments, sgRNAs are contemplated that comprise any one of SEQ ID Nos: 228 to 332, including modifications of Table 4. In some embodiments, sgRNAs are contemplated that comprise any one of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329, including modifications of Table 4. In some embodiments, sgRNAs are contemplated that comprise SEQ ID No: 240. In some embodiments, sgRNAs are contemplated that comprise SEQ ID No: 240, including modifications of Table 4. In some embodiments, sgRNAs are contemplated that comprise SEQ ID No: 242. In some embodiments, sgRNAs are contemplated that comprise SEQ ID No: 358. In other embodiments, the sgRNA comprises a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329, wherein the modification at each nucleotide of the sgRNA corresponding to the nucleotide of the reference sequence identifier in Table 4 is the same or equivalent to the modification shown in the reference sequence identifier in Table 4, optionally further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end, and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end. In some embodiments, the sgRNA further comprises at least three PS bonds connecting nucleotides in the hairpin 1 region. In some embodiments, the sgRNA further comprises at least three PS bonds connecting nucleotides in the hairpin 2 region. In some embodiments, the sgRNA further comprises at least three PS bonds connecting nucleotides in the upper stem region. In some embodiments, the sgRNA forms a ribonucleoprotein complex with S. pyogenes Cas9. BRIEF DESCRIPTION OF THE DRAWINGS

[0143] Figure 1 Shown is the % editing measured by next generation sequencing (NGS) of the mouse transthyretin (TTR) gene after transfection of Neuro2A cells with modified crRNA together with Cas9 mRNA and unmodified trRNA (TR000002).

[0144] Figure 2 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with modified trRNA together with unmodified crRNA (CR000686) and Cas9 mRNA.

[0145] Figure 3 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with Cas9 mRNA and crRNA and trRNA with GC pairings not found in the parental sequence.

[0146] Figure 4 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with modified crRNA, trRNA along with Cas9 mRNA. Standard deviation follows the values.

[0147] Figure 5 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with modified sgRNA together with Cas9 mRNA.

[0148] Figure 6 Figure 2 shows the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with modified crRNA, unmodified trRNA (TR000002) together with Cas9 mRNA. Asterisks indicate dual guides that did not show activity in this experiment for technical reasons. Fig. 9 The dual guide was tested again in the experiment represented in , in which it showed editing activity.

[0149] Figure 7 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with unmodified crRNA (CR000686), modified trRNA along with Cas9 mRNA.

[0150] Figure 8 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with Cas9 mRNA, crRNA, and trRNA pairs with GC pairs or GU mismatches not found in the parental sequence.

[0151] Fig. 9 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neur02A cells with modified crRNA, modified trRNA along with Cas9 mRNA. Standard deviation follows the value.

[0152] Fig.10 Shown is the % editing measured by NGS of the mouse TTR gene after transfection of Neuro2A cells with modified sgRNA together with Cas9 mRNA.

[0153] Fig.11 Shown is the % editing measured by NGS of the mouse Factor VII (FVII) gene after transfection of Neuro2A cells with modified sgRNA together with Cas9 mRNA.

[0154] Fig. 12A and 12B It is shown that after Neuro2A cells were transfected with modified crRNA and unmodified trRNA together with Cas9 mRNA, the expression of Cas9 mRNA in the mouse TI'R ( Fig. 12A ) or FVII( Fig. 12B ) by NGS.

[0155] Fig.13A and 13B Figure 3 shows that after Neuro2A cells were transfected with modified trRNA and unmodified crRNA together with Cas9 mRNA, the expression of mouse TTR ( Fig.13A ) or FVII( Fig. 13B ) by NGS.

[0156] Fig.14A , 14B , 14C and 14D show the levels of interferon α (IFN-α, 14A), interleukin 6 (IL-6, 14B), monocyte chemotactic protein 1 (monocyte chemotactic protein 1, MCP-1, 14C) and tumor necrosis factor α (TNF-α, 14D) in serum after in vivo administration of LNPs containing Cas9 mRNA and sgRNA.

[0157] Fig.15A , 15B and 15C show the in vivo results after administration of LNPs containing Cas9 mRNA and sgRNA. Fig.15A The percentage of total editing in the liver is shown. Fig. 15B Serum TTR levels are shown. Fig. 15C show Fig.15A The mean and standard deviation of the results. Fig.15D Modifications to G000209 sgRNA (SEQ ID NO: 228) are summarized. Figure 1 5E summarizes the modifications to G000267 sgRNA (SEQ ID NO: 234). Fig.15D and 15E In the figure, the nucleotides in bold are modified with 2'-O-Me.

[0158] Fig.16A , 16B, 16C and 16D show the levels of interferon α (IFN-α, 16A), tumor necrosis factor α (TNF-α, 16B), interleukin 6 (IL-6, 16C), and monocyte chemoattractant protein 1 (MCP-1, 16D) in serum after in vivo administration of LNPs containing Cas9 mRNA and sgRNA.

[0159] Fig.17A , 17B , 17C and 17D show the in vivo results after administration of LNPs containing Cas9 mRNA and sgRNA. Fig.17A The percentage of total editing in the liver is shown. Fig. 17B show Fig.17A The mean and standard deviation of the results. Fig. 17C Serum TTR levels are shown. Fig.17D show Fig. 17B The mean and standard deviation of the results.

[0160] Fig.18A , 18B and 18C show the in vivo results after administration of LNPs containing Cas9 mRNA and sgRNA. Fig.18A The percentage of total editing in the liver is shown. Fig.18B Summarize liver editing data. Fig.18C Serum TTR levels are shown. MPK = mg / kg; BLOD = below detection.

[0161] Fig.19A , 19B , 19C and 19D show the levels of interferon α (IFN-α, 19A), monocyte chemoattractant protein 1 (MCP-1, 19B), interleukin 6 (IL-6, 19C), and tumor necrosis factor α (TNF-α, 19D) in serum after in vivo administration of LNPs containing Cas9 mRNA and sgRNA.

[0162] Fig. 20A and 20B It was shown that after in vivo administration of LNPs containing Cas9 mRNA and sgRNA, the FVII locus ( Fig. 20A ) and TTR locus ( Fig. 20B ) in the liver.

[0163] Fig.21A , 21B and 21C shows the annotated sgRNA (SEQ ID NO: 341) ( Fig.21A ), unannotated dgRNA CR000686 (SEQ ID NO: 1) and TR000002 (SEQ ID NO: 188) ( Fig.21B), annotated dgRNA CR000686 (SEQ ID NO: 1) and TR000002 (SEQ ID NO: 188) ( Fig. 21C ) schematic diagram.

[0164] Fig.22A , 22B and 22C show the in vivo results after administration of LNPs containing Cas9 mRNA and sgRNA. Fig.22A The percentage of total editing of the TTR locus in liver is shown. Fig. 22B Summarize liver editing data. Fig. 22C Shows serum TT'R levels.

[0165] Fig.23A , 23B and 23C show the in vivo results after administration of LNPs containing Cas9 mRNA and sgRNA. Fig.23A The percentage of total editing of the TTR locus in liver is shown. Fig. 23B Summarize liver editing data. Fig.23C Serum TTR levels are shown.

[0166] Fig.24A , 24B FIG24A shows the percentage of editing of the total editing of the TTR locus. FIG24B shows the normalized transformation of the percentage of editing as a function of mRNA dose used to calculate EC50. Fig.24C EC50 values ​​are shown for the LNPs tested. DETAILED DESCRIPTION OF THE INVENTION

[0168] Provided herein are modified guide RNAs for gene editing methods, including dual guide RNAs and single guide RNAs. The modified guides are more stable than their unmodified counterparts and show improved in vitro and in vivo efficacy. The sequences of the modified and tested guide RNAs are shown in

[0169] Table 4.

[0170]

[0171]

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[0181]

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[0209]

[0210] "Guide RNA" and "gRNA" are used interchangeably herein to refer collectively to sgRNA, trRNA (also known as tracrRNA), or crRNA (also known as CRISPR RNA). CrRNA and trRNA can be combined on one RNA molecule (single guide RNA [sgRNA]) or as two separate RNA molecules (dual guide RNA [dgRNA]). "Guide RNA" or "gRNA" refers to each type.

[0211] The trRNA sequence may be naturally occurring, or the trRNA sequence may include modifications or changes compared to a naturally occurring sequence.

[0212] As used herein, "editing efficiency" or "editing percentage" or "editing %" is the total number of sequence reads with inserted or deleted nucleotides into the target region domain of interest compared to the total number of sequence reads after passing through the Cas RNP.

[0213] As used herein, "hairpin" describes a nucleic acid loop that is created when a nucleic acid strand folds and forms a base pair with another portion of the same strand. A hairpin may form a structure that includes a loop or U-shape. In some embodiments, a hairpin may consist of an RNA loop. A hairpin may be formed by two complementary sequences that are bound together in a single nucleic acid molecule, with folding or wrinkling of the molecule. In some embodiments, a hairpin comprises a stem or a stem-loop structure.

[0214] As used herein, "region (domain)" describes a conserved nucleic acid group. A region may also be referred to as a "module" or "domain". A region of a gRNA may perform a specific function, such as directing the endonuclease activity of an RNP, as described, for example, in Briner AE et al., Molecular Cell 56: 333-339 (2014). The regions of a gRNA are described in Tables 1-3.

[0215] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" describes a gRNA, for example, together with a nuclease, such as a Cas protein. In some embodiments, the RNP comprises Cas9 and a gRNA.

[0216] As used herein, "stem loop" describes the secondary structure of nucleotides that form a base-paired "stem" that ends in a loop of unpaired nucleic acids. Two regions with the same nucleic acid chain can form a stem when the sequences are at least partially complementary when read in opposite directions. As used herein, a "loop" describes a region of non-base-paired (i.e., non-complementary) nucleotides that may cover the stem. A "tetranucleotide loop" describes a loop of 4 nucleotides. As used herein, the upper stem of an sgRNA may comprise a tetranucleotide loop.

[0217] In certain embodiments involving dgRNA, the "stem" region as used herein describes the secondary structure of nucleotides that form a base pairing region between certain regions of crRNA and trRNA (e.g., the lower and upper stem regions of each RNA). The "stem" region of a dgRNA may also be referred to as a "flagpole" region in the art.

[0218] As used herein, "treatment" encompasses any administration or application of a therapeutic stimulus for a disease to a subject, and includes inhibiting the disease, arresting its progression, alleviating one or more symptoms of the disease, curing the disease, or preventing the recurrence of one or more symptoms of the disease.

[0219] 1. Modification type

[0220] A. 2'-O-methyl modification

[0221] It is believed that the modified sugar controls the rugosity of the nucleotide sugar ring, which is a physical property that affects the binding affinity of the oligonucleotide for the complementary strand, duplex formation, and interaction with nucleases. Substitutions on the sugar ring can thus alter the alignment and rugosity of these sugars. For example, a 2'-O-methyl (2'-O-Me) modification can increase the binding affinity and nuclease stability of an oligonucleotide, although as shown in the Examples, the effect of any modification at a given position in an oligonucleotide needs to be determined empirically.

[0222] The terms "mA", "mC", "mU" or "mG" are used to indicate a nucleotide that has been 2'-O-Me modified.

[0223] The modification of ribonucleotides in the form of 2'-O-methyl ribonucleotides can be depicted as follows:

[0224]

[0225] B. 2'-O-(2-methoxyethyl) modification

[0226] In some embodiments, the modification may be 2'-O-(2-methoxyethyl) (2'-O-moe). The modification of a ribonucleotide in the form of a 2'-O-moe ribonucleotide may be depicted as follows:

[0227]

[0228] The terms "moeA", "moeC", "moeU" or "moeG" are used to indicate a nucleotide that has been 2'-.O-moe modified.

[0229] C.2'-Fluoro modification

[0230] Another chemical modification that has been shown to affect the nucleotide sugar ring is halogen substitution. For example, 2'-fluoro (2'-F) substitutions on the nucleotide sugar ring can increase oligonucleotide binding affinity and nuclease stability.

[0231] In the present application, the terms "fA", "fC", "fU" or "fG" are used to indicate a nucleotide that has been substituted with 2'-F.

[0232] The substitution of 2'-F can be depicted as follows:

[0233]

[0234] D. Phosphorothioate modification

[0235] A phosphorothioate (PS) linkage or bond refers to a sulfur-substituted phosphodiester linkage, such as a bond between nucleotide bases that replaces one of the non-bridging phosphate oxygens. When phosphorothioates are used to produce oligonucleotides, the modified oligonucleotide may also be referred to as an S-oligonucleotide.

[0236] "*" is used to depict PS modification. In this application, the terms A*, C*, U* or G* are used to indicate the nucleotide that is linked to the next (eg, 3') nucleotide with a PS bond.

[0237] In the present application, the term "mA*", "mC*", "mU*" or "mG*" is used to indicate a nucleotide that has been substituted with 2'-O-Me and is linked to the next (e.g., 3') nucleotide with a PS bond. Similarly, the term "fA*", "f℃*", "fU*" or "fG*" is used to indicate a nucleotide that has been substituted with 2'-F and is linked to the next (e.g., 3') nucleotide with a PS bond. The embodiments described herein encompass equivalents of PS connections or bonds.

[0238] The following diagram shows the substitution of an S- to a non-bridging phosphate oxygen, creating a PS bond in place of the phosphodiester bond:

[0239]

[0240] EG-C substitution

[0241] In some embodiments, gRNA is modified by sequence substitution without chemical modification. In some embodiments, the modified gRNA is transformed with GC pairing (e.g., in the lower and / or upper stem region), which is not found in the parental gRNA sequence. In some embodiments, the modified gRNA is transformed by GU mispairing ("GU wobbles" or mispairing pairing) not found in the parental gRNA sequence.

[0242] F. Reverse abasic modification

[0243] Abasic nucleotides refer to those nucleotides that lack a nitrogenous base. The following figure depicts an oligonucleotide with an abasic (also called apurinic) site that lacks a base:

[0244]

[0245] Inverted bases are those bases that have a bond that is inverted from the normal 5' to 3' bond (i.e., a 5' to 5' bond or a 3' to 3' bond). For example:

[0246]

[0247] Abasic nucleotides can be connected to reverse bonds. For example, abasic nucleotides can be connected to terminal 5' nucleotides via a 5' to 5' bond, or abasic nucleotides can be connected to terminal 3' nucleotides via a 3' to 3' bond. Reverse abasic nucleotides at terminal 5' or 3' nucleotides can also be referred to as reverse abasic end caps. In the present application, the term "invd" indicates reverse abasic nucleotide bonds.

[0248] Modifications of the above and their equivalents are included within the scope of the embodiments described herein.

[0249] 2. Guide RNA Composition

[0250] The present invention encompasses compositions comprising guide RNA. In some embodiments, the guide RNA comprises trRNA. In some embodiments, the guide RNA comprises crRNA. In some embodiments, the guide RNA comprises crRNA and trRNA. In some embodiments, the guide RNA comprises crRNA and trRNA as sgRNA on one RNA molecule. In some embodiments, the guide RNA comprises crRNA and trRNA as dgRNA on two RNA molecules. In dgRNA, the two RNA molecules can associate via base pairing.

[0251] In some embodiments, the guide RNA comprises a 5' terminal region. In some embodiments, the guide RNA does not comprise a 5' terminal region. In some embodiments, the 5' terminal region comprises a "spacer" region as described in BrinerAE et al., Molecular Cell 56: 333-339 (2014) for sgRNA (but applicable to all guide RNAs herein). In some embodiments, the 5' terminal region comprises a 5' end modification. The 5' terminal region with or without a spacer can be combined with crRNA, trRNA, sgRNA and / or dgRNA. The spacer is sometimes also referred to as a "guidance region", "guidance domain" or "target domain" herein and by other documents. As used herein, "target sequence" refers to a nucleic acid sequence that guides the cutting of a nuclease by a guide region / domain. In some embodiments, the spyCas9 protein can be directed to the target sequence of a target nucleic acid molecule by the guide region / domain through the nucleotides present in the spacer. In some embodiments, the guide RNA does not comprise a spacer.

[0252] In some embodiments, the guide RNA described herein comprises or consists of any of the sequences shown in Table 4. However, it should be noted that when a sequence shows a guide / spacer region, it should be recognized that the composition may or may not contain this region. In addition, guide RNAs are contemplated that include modifications of any of the sequences shown in Table 4 and identified 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 the modification pattern of the guide sequence of Table 4. The modification pattern includes the relative positioning and identity of the modifications of the gRNA or gRNA region (e.g., 5' terminal region, lower stem region, protrusion region, upper stem region, connecting region, hairpin 1 region, hairpin 2 region, 3' terminal region). In some embodiments, the modification pattern contains modifications of any of the sequences shown in the sequence column of Table 4, or at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% of the modifications of one or more regions of the sequence. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the modification pattern of any of the sequences shown in the sequence column of Table 4. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to one or more regions of the sequences shown in Table 4, such as the 5' terminal region, the lower stem region, the protuberance region, the upper stem region, the connecting region, the hairpin 1 region, the hairpin 2 region, and / or the 3' terminal region. For example, in some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the modification pattern of the sequence through the 5' terminal region. In some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the lower stem region. In some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the protuberance region. In some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the upper stem region. In some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the linker region.In some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the hairpin 1 region. In some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the hairpin 2 region. In some embodiments, guide RNAs are contemplated wherein the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the 3' terminal region. In some embodiments, the modification pattern differs from the sequence of Table 4 at 0, 1, 2, 3, 4, 5, or 6 nucleotides, or a modification pattern of a region of such a sequence (e.g., 5' end, lower stem, protrusion, upper stem, junction, hairpin 1, hairpin 2, 3' end). In some embodiments, the gRNA comprises a modification that differs from the modification of the sequence of Table 4 at 0, 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, the gRNA comprises a modification that differs from the modification of the sequence of Table 4 at 0, 1, 2, 3, 4, 5, or 6 nucleotides.

[0253] In some embodiments, the gRNA comprises 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the gRNA comprises 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides. In some embodiments, the gRNA comprises 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the gRNA comprises phosphorothioate (PS) bonds between nucleotides.

[0254] In some embodiments, the gRNA comprises a 5' end modification, a 3' end modification, or a 5' and 3' end modification. In some embodiments, the 5' end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the 5' end modification comprises a 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe) and / or 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the 5' end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe) and / or 2'-fluoro (2'-F) modified nucleotide. The (end) end modification may comprise a phosphorothioate (PS), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe) and / or 2'-fluoro (2'-F) modified nucleotide. The embodiments described herein also encompass equivalent (terminal) terminal modifications. In some embodiments, the gRNA comprises a (terminal) terminal modification in combination with a modification of one or more regions of the gRNA.

[0255] A. Composition of sgRNA

[0256] In some embodiments, the compositions and methods of the present invention include gRNA, which includes crRNA and trRNA that guide a nuclease such as Cas9 to a target DNA sequence. In some embodiments, the gRNA described herein can be combined on one RNA molecule (single guide RNA or sgRNA).

[0257] In some embodiments, the present invention comprises an sgRNA comprising or consisting of any one of the sequences set forth in SEQ ID Nos: 228 to 332.

[0258] In some embodiments, a sgRNA comprising any of the modified sequences of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329 is provided. In some embodiments, a sgRNA comprising any of the modified sequences of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329 is contemplated, wherein the sgRNA further comprises a 5' "spacer" sequence ("guide sequence") that is complementary to the target sequence and guides Cas9 to its target for cleavage. In some cases, the invention comprises a sgRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to a nucleic acid of any of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier.

[0259] 1. Domain structure of sgRNA

[0260] Briner AE et al., Molecular Cell 56:333-339 (2014) describe functional domains of sgRNAs referred to herein as "domains", including a "spacer" domain responsible for targeting, a "lower stem", a "protuberance", an "upper stem" (which may include a tetranucleotide loop), a "linker", and "hairpin 1" and "hairpin 2" domains. See Briner et al., p. 334, Figure 1 A.

[0261] Table 1 and Fig.21A A description of the domains of sgRNAs as used herein is provided. In Table 1, "n" between regions represents a variable number of nucleotides, such as 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some embodiments, n is equal to 0. In some embodiments, n is equal to 1.

[0262] Table 1: Regions of sgRNA (linear view, 5' to 3')

[0263] LS1-6 B1-2 US1-12 B3-6 5′ terminal region (n) lower stem area n Carina n upper stem area n Carina n

[0264] (Continued below)

[0265] LS7-12 N1-18 H1-1 to H1-12 H2-1 to H2-15 lower stem area n Connection Area n Hairpin Zone 1 n Hairpin Zone 2 3' terminal region

[0266] a) 5' terminal region

[0267] In some embodiments, the sgRNA comprises nucleotides at the 5' end as shown in Table 1. In some embodiments, the 5' end of the sgRNA comprises a spacer or guide region for guiding the Cas protein to the target nucleotide sequence. In some embodiments, the 5' end does not comprise a spacer or guide region. In some embodiments, the 5' end comprises a spacer and other nucleotides that are not used to guide the Cas protein to the target nucleotide region.

[0268] In some embodiments, the guide region comprises the first 1 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at the 5' end of the sgRNA. In some embodiments, the guide region comprises 20 nucleotides. In some embodiments, the guide region may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more 25 nucleotides. In some embodiments, the guide region may comprise 17 nucleotides. In some embodiments, the guide region may comprise 18 nucleotides. In some embodiments, the guide region may comprise 19 nucleotides.

[0269] In some embodiments, the selection of the guide region is determined based on the target sequence within the gene of interest for editing. For example, in some embodiments, the sgRNA comprises a guide region that is complementary to the target sequence of the gene of interest.

[0270] In some embodiments, the target sequence in the gene of interest may be complementary to the guide region of the sgRNA. In some embodiments, the degree of complementarity or identity between the guide region of the sgRNA and its corresponding target sequence in the gene of interest may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the guide region of the sgRNA and the target region domain of the gene of interest may be 100% complementary or identical. In other embodiments, the guide region of the sgRNA and the target region domain of the gene of interest may contain at least one mismatch. For example, the guide region of the sgRNA and the target sequence of the gene of interest may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches, wherein the total length of the target sequence is at least about 17, 18, 19, 20 base pairs or more than 20 base pairs. In some embodiments, the guide region of the sgRNA and the target region domain of the target gene may contain 1 to 6 mismatches, wherein the guide sequence comprises at least about 17, 18, 19, 20 nucleotides, or more than 20 nucleotides. In some embodiments, the guide region of the sgRNA and the target region domain of the target gene may contain 1, 2, 3, 4, 5, or 6 mismatches, wherein the guide sequence comprises about 20 nucleotides. The 5' end may include nucleotides that are not considered as the guide region (i.e., not used to guide the cas9 protein to the target nucleic acid).

[0271] b) Lower stem area

[0272] In some embodiments, the sgRNA comprises a lower stem (LS) region separated by a bulge region and an upper stem region when viewed linearly. See Table 1.

[0273] In some embodiments, the lower stem region comprises 1 to 12 nucleotides, for example, in one embodiment, the lower stem region comprises LS1 to LS12. In some embodiments, the lower stem region comprises a ratio of Table 1 and Fig.21A In some embodiments, the lower stem region comprises fewer nucleotides than those shown in Tables 1 and Fig.21A When the lower stem region contains more nucleotides than those shown in Table 1 and Fig.21A When fewer or more nucleotides are shown in the schematic diagram, the modification pattern should be maintained as is apparent to those skilled in the art.

[0274] In some embodiments, when read in the opposite direction, the lower stem region has nucleotides with complementary nucleic acid sequences. In some embodiments, the complementarity in the nucleic acid sequence of the lower stem region produces a secondary structure of the stem in the sgRNA (e.g., the region may base pair with each other). In some embodiments, when read in the opposite direction, the lower stem regions may not be completely complementary to each other.

[0275] c) Protrusion area

[0276] In some embodiments, the sgRNA comprises a protuberance region comprising six nucleotides B1 to B6. When viewed linearly, the protuberance region is divided into two regions. See Table 1. In some embodiments, the protuberance region comprises six nucleotides, wherein the first two nucleotides are followed by an upper stem region, followed by four nucleotides after the protuberance region. In some embodiments, the protuberance region comprises a ratio of nucleotides in Table 1 and Fig.21A In some embodiments, the protrusion region comprises fewer nucleotides than those shown in Tables 1 and Fig.21A When the bulge region contains more nucleotides than those shown in Table 1 and Fig.21A When fewer or more nucleotides are shown in the schematic diagram, the modification pattern should be maintained as is apparent to those skilled in the art.

[0277] In some embodiments, the presence of the protrusion region results in a directional kink between the upper and lower stem modules in the sgRNA.

[0278] d) Upper stem region

[0279] In some embodiments, the sgRNA comprises an upper stem region comprising 12 nucleotides. In some embodiments, the upper stem region comprises a loop sequence. In some cases, the loop is a tetranucleotide loop (a loop consisting of four nucleotides).

[0280] In some embodiments, the upper stem region comprises a ratio of Table 1 and Fig.21A In some embodiments, the upper stem region comprises fewer nucleotides than those shown in Tables 1 and Fig.21A When the upper stem region contains more nucleotides than those shown in Table 1 and Fig.21AWhen fewer or more nucleotides are shown in the schematic diagram, the modification pattern should be maintained as is apparent to those skilled in the art.

[0281] In some embodiments, when read in the opposite direction, the upper stem region has nucleotides that are complementary to the nucleic acid sequence. In some embodiments, the complementarity in the nucleic acid sequence of the upper stem region produces a secondary structure of the stem in the sgRNA (e.g., the region may base pair with each other). In some embodiments, when read in the opposite direction, the upper stem regions may not be completely complementary to each other.

[0282] e) Connection area

[0283] In some embodiments, the sgRNA comprises a linker region between the lower stem region and the hairpin 1 region. In some embodiments, the linker region comprises 18 nucleotides. In some embodiments, the linker region comprises as shown in Table 1 and Fig.21A Nucleotides N1 to N18 are shown in FIG.

[0284] In some embodiments, the connecting region comprises a ratio of Table 1 and Fig.21A In some embodiments, the connecting region comprises fewer nucleotides than those shown in Tables 1 and Fig.21A When the junction region contains more nucleotides than those shown in Table 1 and Fig.21A When fewer or more nucleotides are shown in the schematic diagram, the modification pattern should be maintained as is apparent to those skilled in the art.

[0285] In some embodiments, the connecting region has nucleotides that are complementary to the nucleic acid sequence when read in the opposite direction. In some embodiments, the complementarity in the nucleic acid sequence generates a secondary structure of the stem and / or stem loop in the sgRNA (e.g., certain nucleotides in the connecting region may base pair with each other). In some embodiments, the connecting regions may not be completely complementary to each other when read in the opposite direction.

[0286] f) Hairpin area

[0287] In some embodiments, the sgRNA comprises one or more hairpin regions. In some embodiments, the hairpin region is downstream of the connecting region (e.g., 3' relative to it). In some embodiments, the region of nucleotides immediately downstream of the connecting region is referred to as "hairpin 1 region" or "H1". In some embodiments, the region of nucleotides 3' relative to hairpin 1 is referred to as "hairpin 2 region" or "H2". In some embodiments, the hairpin region comprises hairpin 1 region and hairpin 2 region. In some embodiments, the sgRNA comprises only hairpin 1 region or hairpin 2 region.

[0288] In some embodiments, the hairpin 1 region comprises 12 nucleic acids immediately downstream of the connecting region. In some embodiments, the hairpin 1 region comprises the nucleic acids shown in Table 1 and Fig.21ANucleotides H1-1 to H1-12 are shown in FIG.

[0289] In some embodiments, the hairpin 2 region comprises 15 nucleic acids downstream of the hairpin 1 region. In some embodiments, the hairpin 2 region comprises the nucleic acids shown in Tables 1 and Fig.21A Nucleotides H2-1 to H2-15 are shown in FIG.

[0290] In some embodiments, one or more nucleotides are present between hairpin 1 region and hairpin 2 region. One or more nucleotides between hairpin 1 region and hairpin 2 region may be modified or unmodified. In some embodiments, hairpin 1 region and hairpin 2 region are separated by one nucleotide. In some embodiments, the hairpin region comprises a ratio of Table 1 and Fig.21A In some embodiments, the hairpin region comprises fewer nucleotides than those shown in Tables 1 and Fig.21A When the hairpin region contains more nucleotides than those shown in Table 1 and Fig.21A When fewer or more nucleotides are shown in the schematic diagram, the modification pattern should be maintained as is apparent to those skilled in the art.

[0291] In some embodiments, when read in the opposite direction, the hairpin region has nucleotides that are complementary to the nucleic acid sequence. In some embodiments, when read in the opposite direction, the hairpin region may not be completely complementary to each other (e.g., the top or loop of the hairpin region contains unpaired nucleotides).

[0292] In some embodiments, the sgRNA comprises a hairpin 1 region substituted with "n" nucleotides, wherein "n" is an integer between 1 and 50, 40, 30, 20, 15, 10, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of the sgRNA is substituted with 2 nucleotides.

[0293] g) 3' terminal region

[0294] In some embodiments, the sgRNA comprises nucleotides after the hairpin region. In some embodiments, the 3' terminal region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nucleotides or more than 20 nucleotides, for example, does not bind to the secondary structure of the hairpin region. In some embodiments, the 3' terminal region comprises 1, 2, 3 or 4 nucleotides that do not bind to the secondary structure of the hairpin region. In some embodiments, the 3' terminal region comprises 4 nucleotides that do not bind to the secondary structure of the hairpin region. In some embodiments, the 3' terminal region comprises 1, 2 or 3 nucleotides that do not bind to the secondary structure of the hairpin region.

[0295] 2. Modification of sgRNA

[0296] In some embodiments, the invention comprises an sgRNA comprising one or more modifications in one or more of the following regions: nucleotides at the 5' end; lower stem region; protuberance region; upper stem region; connecting region; hairpin 1 region; hairpin 2 region; and nucleotides at the 3' end.

[0297] In some embodiments, the modification comprises 2'-o-methyl (2'-O-Me) modified nucleotides. In some embodiments, the modification comprises 2'-OO-(2-methoxyethyl) (2'-O-moe) modified nucleotides. In some embodiments, the modification comprises 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the modification comprises phosphorothioate (PS) bonds between nucleotides.

[0298] In some embodiments, the sgRNA comprises a modification of 1, 2, 3, or 4 of the first 4 nucleotides at its 5' end. In some embodiments, the first three or four nucleotides at the 5' end, and the last three or four nucleotides at the 3' end are modified. In some embodiments, the first four nucleotides at the 5' end, and the last four nucleotides at the 3' end are linked to a phosphorothioate (PS) bond. In some embodiments, the modification comprises 2'-O-Me. In some embodiments, the modification comprises 2'-F. In some embodiments, the modification comprises 2'-O-moe.

[0299] In some embodiments, the sgRNA comprises a modification at 1, 2, 3, or 4 of the first 4 nucleotides at the 5' end. In some embodiments, the sgRNA comprises a modification at 1, 2, 3, or 4 of the first 4 nucleotides at the 3' end. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a PS bond, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2'-O-Me or 2'-O-moe modification.

[0300] In some embodiments, the first four nucleotides at the 5' terminus and the last four nucleotides at the 3' terminus are linked to PS bonds, and the first three nucleotides at the 5' terminus and the last three nucleotides at the 3' terminus comprise a 2'-F modification.

[0301] In some embodiments, sgRNAs are provided wherein LS1, LS6, LS7, LS8, LS11, and LS12 are modified with 2'-O-Me. In some embodiments, each of the nucleotides in the protuberance region of the sgRNA is modified with 2'-O-Me. In some embodiments, each of the nucleotides in the upper stem region of the sgRNA is modified with 2'-O-Me. In some embodiments, N16, N17, and N18 in the linking region of the sgRNA are modified with 2'-O-Me. In some embodiments, each of the nucleotides in the hairpin 1 region of the sgRNA is modified with 2'-O-Me. In some embodiments, each of the nucleotides in the hairpin 2 region of the sgRNA is modified with 2'-O-Me.

[0302] In some embodiments, the sgRNA comprises 2'-O-Me modified nucleotides at the following nucleotides: the first three nucleotides at the 5' end; LS1, LS6, LS7, LS8, LS11, and LS12; B1 and B2 in the bulge region; each of the nucleotides in the upper stem region of the sgRNA; N16, N17, and N18 in the linker region; each of the nucleotides in the hairpin 1 region; each of the nucleotides in the hairpin 2 region; and the last four nucleotides at the 3' end.

[0303] In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises a 2'-O-Me or 2'-F modified nucleic acid at the first three nucleotides at the 5' end, and a 2'-O-Me or 2'-F modified nucleic acid at the last four nucleotides at the 3' end. In some embodiments, LS9 and LS10 are modified with 2'-F. In some embodiments, N15, N16, N17, and N18 are modified with 2'-F. In some embodiments, H2-9, H2-10, H2-11, H2-12, H2-13, HS-14, and H2-15 are modified with 2'-F. In some embodiments, the second to last, third to last, and fourth to last nucleotides at the 3' end are modified with 2'-F.

[0304] In some embodiments, a single guide RNA (sgRNA) is provided that comprises a 2'-F modified nucleic acid at the following nucleotides: LS9 and LS10 in the lower stem region; N15, N16, N17, and N18 in the linker region; and H2-9, H2-10, H2-11, H2-12, H2-13, HS-14, and H2-15 in the hairpin 2 region. In some embodiments, the sgRNA further comprises a 2'-F modified nucleotide at the second to last, third to last, and fourth to last nucleotide at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises a 2'-O-Me or 2'-F modified nucleic acid at the first three nucleotides at the 5' end, and a 2'-O-Me or 2'-F modified nucleic acid at three of the last four nucleotides at the 3' end.

[0305] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-O-Me modified nucleotides at LS1 and LS6; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2.1 to H2-15; and 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0306] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-F modified nucleotides at LS1 to LS6; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides at "n" between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2-1 to H2-15; and 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0307] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-F modified nucleotides at LS2 to LS5; 2'-O-Me modified nucleotides at LS1 and LS6; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides at "n" between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2-1 to H2-15; and 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0308] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at LS7, LS8, LS11, and LS12; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides at "n" between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2-1 to H2-15; and 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0309] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at LS8, LS10 and LS12; 2'-OF modified nucleotides at LS7, LS9 and LS11; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2-1 to H2-15; and 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0310] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS1 1, and LS12; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2-1 to H2-15; and 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0311] In some embodiments, a single guide RNA (sgRNA) is provided, which includes 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and LS12; 2'-F modified nucleotides at LS9 and LS10; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2-1 to H2-15; and 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. In some embodiments, the sgRNA further includes three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0312] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at H1-1 to H1-12; 2'-O-Me modified nucleotides between the hairpin 1 region and the hairpin 2 region; 2'-O-Me modified nucleotides at H2-1 to H2-8; 2'-F modified nucleotides at H2-9 to H2-15; 2'-F modified nucleotides at the second to last, third to last, and fourth to last nucleotides at the 3' end; and 2'-O-Me modified nucleotides at the last nucleotide at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0313] In some embodiments, a single guide RNA (sgRNA) is provided, which comprises 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; 2'-O-Me modified nucleotides at US1 to US12; 2'-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12; 2'-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11; 2'-F modified nucleotides between hairpin 1 region and hairpin 2 region; -F modified nucleotides; 2'-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14; 2'-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15; 2'-F modified nucleotides at the second to last and fourth to last nucleotides at the 3' terminus; and 2'-O-Me modified nucleotides at the third to last and last nucleotides at the 3' terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' terminus and three PS bonds connecting the last four nucleotides at the 3' terminus.

[0314] In some embodiments, disclosed herein are single guide RNAs (sgRNAs) comprising 2'-O-Me modifications at nucleotides LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15; and 2'-F modifications at LS7, LS9, LS11; H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises 2'-O-Me modified nucleotides at the last and third-to-last nucleotides at the 3' terminus; and 2'-F modified nucleotides at the second-to-last and third-to-last nucleotides at the 3' terminus.

[0315] In some embodiments, disclosed herein is an sgRNA comprising a nucleic acid of any one of SEQ ID Nos: 228 to 232. In some embodiments, disclosed herein is an sgRNA comprising a nucleic acid of any one of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329. In some embodiments, disclosed herein is an sgRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to a nucleic acid of any one of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0316] In some embodiments, an sgRNA is provided that comprises a 5' end modification and one or more modifications of one or more of: an upper stem region; a hairpin 1 region; and a hairpin 2 region, wherein the 5' end modification comprises at least two phosphorothioate bonds within seven nucleotides before the 5' end.

[0317] In some embodiments, an sgRNA is provided that comprises a 5' end modification and one or more modifications of one or more of the following: an upper stem region; a hairpin 1 region; and a hairpin 2 region, wherein the 5' end modification comprises one or more phosphorothioate bonds at the 5' end of the RNA. In some embodiments, the one or more phosphorothioate bonds connect the 5' terminal nucleotides.

[0318] In some embodiments, an sgRNA is provided that comprises a 5' end modification and one or more modifications of one or more of the following: an upper stem region; a hairpin 1 region v, and a hairpin 2 region, wherein the 5' end modification comprises one or more phosphorothioate bonds within seven nucleotides before the 5' end.

[0319] In some embodiments, a sgRNA is provided that comprises any one of the modified sgRNA sequences of SEQ ID Nos: 228 to 332.

[0320] In some embodiments, a sgRNA is provided that comprises or consists of any one of the modified sgRNA sequences of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329.

[0321] In some embodiments, the present invention comprises an sgRNA comprising any one of the modified sequences of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329, wherein the sgRNA further comprises a 5' spacer sequence that is at least partially complementary to the target sequence and guides Cas9 to its target for cleavage.

[0322] In some embodiments, the invention comprises an sgRNA comprising nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleotides of any one of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier. That is, the nucleotides A, U, C, and G may differ by 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% compared to those shown in the sequence, but the modifications remain the same.

[0323] In some embodiments, the invention comprises an sgRNA comprising one or more modifications in one or more of the following regions: nucleotides at the 5' end; lower stem region; protuberance region; upper stem region; connecting region; hairpin 1 region; hairpin 2 region; and nucleotides at the 3' end.

[0324] In some embodiments, the modification comprises 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the modification comprises 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the modification comprises phosphorothioate (PS) bonds between nucleotides. In some embodiments, the modification comprises reverse abasic nucleotides.

[0325] In some embodiments, an sgRNA is provided that includes 2'-O-Me modified nucleotides at: the first three nucleotides in the 5' terminal region; LS1, LS6, LS7, LS8, LS11, and LS12 in the lower stem region; B1 and B2 in the bulge region; each of the nucleotides in the upper stem region; N16, N17, and N18 in the connecting region; each of the nucleotides in the hairpin 1 region; one nucleotide between the hairpin 1 region and the hairpin 2 region; each of the nucleotides in the hairpin 2 region; and the last four nucleotides at the 3' end. In one embodiment, the sgRNA further includes three PS bonds between the first four nucleotides at the 5' end and three PS bonds between the last four nucleotides at the 3' end.

[0326] In some embodiments, an sgRNA is provided that includes 2'-O-Me modified nucleotides at: the first three nucleotides in the 5' terminal region; LS1, LS6, LS7, LS8, LS11, and LS12 in the lower stem region; B1 to B6 in the protrusion region; each of the nucleotides in the upper stem region; N16, N17, and N18 in the connecting region; each of the nucleotides in the hairpin 1 region; one nucleotide between the hairpin 1 region and the hairpin 2 region; each of the nucleotides in the hairpin 2 region; and the last four nucleotides at the 3' end. In one embodiment, the sgRNA further includes three PS bonds between the first four nucleotides at the 5' end and three PS bonds between the last four nucleotides at the 3' end.

[0327] In some embodiments, an sgRNA is provided that comprises 2'-F modified nucleotides at: LS9 and LS10 in the lower stem region; 15-N18 in the linker region; H2-9 to HS-15 in the hairpin 2 region; and the second to last, third to last, and fourth to last nucleotides in the 3' terminal region.

[0328] In some embodiments, an sgRNA is provided that comprises 2'-F modified nucleotides at: each nucleotide in the lower stem region; 15-N18 in the linker region; H2-9 to HS-15 in the hairpin 2 region; and the second to last, third to last, and fourth to last nucleotides in the 3' terminal region.

[0329] In some embodiments, a single guide RNA (sgRNA) is provided that comprises 2'-O-Me modified nucleotides at LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, H2-15, and the last and third to last nucleotides at the 3' terminus; and 2'-F modifications at LS7, LS9, LS11; H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, H2-14, and the second to last and fourth to last nucleotides at the 3' terminus.

[0330] The present invention encompasses each of the following embodiments:

[0331] Embodiment 01. A single guide RNA (sgRNA) comprising one or more modifications in one or more of the following regions:

[0332] a.5' end;

[0333] b. Lower stem region;

[0334] c. protrusion area;

[0335] d. Upper stem region v

[0336] e. Connection area;

[0337] f. Hairpin 1 area;

[0338] g. Hairpin 2 area; and

[0339] h.3' end.

[0340] Embodiment 02. The sgRNA of embodiment 1, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0341] Embodiment 03. The sgRNA of Embodiment 1, wherein the modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[0342] Embodiment 04. The sgRNA of embodiment 1, wherein the modification comprises a phosphorothioate (PS) bond between nucleotides.

[0343] Embodiment 05. The sgRNA of any one of Embodiments 1 to 3, wherein the first three or four nucleotides at the 5' end and the last three or four nucleotides at the 3' end are modified.

[0344] Embodiment 06. The sgRNA of any one of Embodiments 1 to 5, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to phosphorothioate (PS) bonds.

[0345] Embodiment 07. The sgRNA of Embodiment 5, wherein the modification comprises 2'-O-Me.

[0346] Embodiment 08. The sgRNA of Embodiment 5, wherein the modification comprises 2'-F.

[0347] Embodiment 09. The sgRNA of any one of embodiments 1 to 7, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a PS bond, and wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2'-O-Me modification.

[0348] Embodiment 10. The sgRNA of any one of embodiments 1 to 8, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a PS bond, and wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2'-F modification.

[0349] Embodiment 11. The sgRNA of any one of embodiments 1 to 10, wherein LS1, LS6, LS7, LS8, LS11 and LS12 are modified with 2'-O-Me.

[0350] Embodiment 12. The sgRNA of any one of embodiments 1 to 11, wherein each of the nucleotides in the protruding region is modified with 2'-O-Me.

[0351] Embodiment 13. The sgRNA of any one of embodiments 1 to 12, wherein each of the nucleotides in the upper stem region is modified with 2'-O-Me.

[0352] Embodiment 14. The sgRNA of any one of embodiments 1 to 13, wherein N16, N17, and N18 in the linker region are modified with 2′-O-Me.

[0353] Embodiment 15. The sgRNA of any one of embodiments 1 to 14, wherein each of the nucleotides in the hairpin 1 region is modified with 2'-O-Me.

[0354] Embodiment 16. The sgRNA of any one of embodiments 1 to 15, wherein each of the nucleotides in the hairpin 2 region is modified with 2'-O-Me.

[0355] Embodiment 17. A single guide RNA (sgRNA) comprising a 2'-O-Me modified nucleic acid at the following nucleotides:

[0356] a. The first three nucleotides at the 5' end;

[0357] b. LS1, LS6, LS7, LS8, LS11 and LS12 in the lower stem region;

[0358] c. B1 and B2 in the protrusion area;

[0359] d. Each nucleotide in the upper stem region;

[0360] e. N16, N17 and N18 in the connection area;

[0361] f. Each nucleotide in the hairpin 1 region;

[0362] g. each nucleotide in region 2 of the hairpin; and

[0363] h. Last four nucleotides at the 3' end.

[0364] Embodiment 18. The sgRNA of embodiment 17, wherein B3 to B6 are modified with 2'-O-Me.

[0365] Embodiment 19. The sgRNA of embodiment 17, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0366] Embodiment 20. The sgRNA of any one of embodiments 1 to 10, wherein LS9 and LS10 are modified with 2'-F.

[0367] Embodiment 21. The sgRNA of any one of embodiments 1 to 10 and 20, wherein N15, N16, N17 and N18 are modified with 2'-F.

[0368] Embodiment 22. The sgRNA of any one of embodiments 1 to 10 and 20 to 21, wherein H2-9, H2-10, H2-11, H2-12, H2-13, H2-14 and H2-15 are modified with 2'-F.

[0369] Embodiment 23. The sgRNA of any one of embodiments 1 to 10 and 21 to 22, wherein the second to last, third to last, and fourth to last nucleotides at the 3' end are modified with 2'-F.

[0370] Embodiment 24. A single guide RNA (sgRNA) comprising a 2'-F modified nucleotide at the following position:

[0371] a. LS9 and LS10 in the lower stem region;

[0372] b. N15, N16, N17 and N18 in the connection region; and

[0373] c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14 and H2-15 in the hairpin 2 region.

[0374] Embodiment 25. The sgRNA of Embodiment 24, further comprising 2'-F modified nucleotides at the second to last, third to last, and fourth to last nucleotides at the 3' end.

[0375] Embodiment 26. The sgRNA of any one of Embodiments 24 or 25, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0376] Embodiment 27. The sgRNA of any one of Embodiments 24 to 26, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' terminus, and 2'-O-Me or 2'-F modified nucleotides at three of the last four nucleotides at the 3' terminus.

[0377] Embodiment 28. A single guide RNA (sgRNA) comprising

[0378] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0379] b. 2'-O-Me modified nucleotides at LS1 and LS6;

[0380] c. 2'-O-Me modified nucleotides at US1 to US12;

[0381] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0382] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0383] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0384] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0385] Embodiment 29. The sgRNA of embodiment 28, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0386] Embodiment 30. A single guide RNA (sgRNA) comprising

[0387] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0388] b. 2'-F modified nucleotides at LS1 to LS6;

[0389] c. 2'-O-Me modified nucleotides at US1 to US12;

[0390] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0391] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0392] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0393] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0394] Embodiment 31. The sgRNA of embodiment 30, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0395] Embodiment 32. A single guide RNA (sgRNA) comprising

[0396] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0397] b. 2'-F modified nucleotides at LS2 to LS5;

[0398] c. 2'-O-Me modified nucleotides at LS1 and LS6;

[0399] d. 2'-O-Me modified nucleotides at US1 to US12;

[0400] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0401] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0402] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0403] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0404] Embodiment 33. The sgRNA of embodiment 32, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0405] Embodiment 34. A single guide RNA (sgRNA) comprising

[0406] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0407] b. 2'-O-Me modified nucleotides at US1 to US12;

[0408] c. 2'-O-Me modified nucleotides at LS7, LS8, LS11 and LS12;

[0409] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0410] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0411] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0412] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0413] Embodiment 35. The sgRNA of embodiment 34, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0414] Embodiment 36. A single guide RNA (sgRNA) comprising

[0415] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0416] b. 2'-O-Me modified nucleotides at US1 to US12;

[0417] c. 2'-O-Me modified nucleotides at LS7, LS8, LS11 and LS12;

[0418] d. 2'-F modified nucleotides at LS9 and LS10;

[0419] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0420] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0421] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0422] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0423] Embodiment 37. The sgRNA of Embodiment 36, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0424] Embodiment 38. A single guide RNA (sgRNA) comprising

[0425] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0426] b. 2'-O-Me modified nucleotides at US1 to US12;

[0427] c. 2'-O-Me modified nucleotides at LS8, LS10 and LS12;

[0428] d. 2'-OF modified nucleotides at LS7, LS9 and LS11;

[0429] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0430] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0431] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0432] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0433] Embodiment 39. The sgRNA of embodiment 32, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0434] Embodiment 40. A single guide RNA (sgRNA) comprising

[0435] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0436] b. 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11 and LS12

[0437] c. 2'-O-Me modified nucleotides at US1 to US12;

[0438] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0439] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0440] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0441] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0442] Embodiment 41. The sgRNA of embodiment 40, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0443] Embodiment 42. A single guide RNA (sgRNA) comprising

[0444] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0445] b. 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11 and LS12;

[0446] c. 2'-F modified nucleotides at LS9 and LS10;

[0447] d. 2'-O-Me modified nucleotides at US1 to US12;

[0448] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0449] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0450] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0451] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end.

[0452] Embodiment 43. The sgRNA of Embodiment 43, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0453] Embodiment 44. A single guide RNA (sgRNA) comprising

[0454] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0455] b. 2'-O-Me modified nucleotides at US1 to US12;

[0456] c. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0457] d. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0458] e. 2'-O-Me modified nucleotides at H2-1 to H2-8;

[0459] f. 2'-F modified nucleotides at H2-9 to H2-15;

[0460] g. 2'-F modified nucleotides at the second to last, third to last and fourth to last nucleotides at the 3' terminus; and

[0461] h. 2'-O-Me modified nucleotide at the last nucleotide at the 3' end.

[0462] Embodiment 45. The sgRNA of Embodiment 44, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0463] Embodiment 46. A single guide RNA (sgRNA) comprising

[0464] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end;

[0465] b. 2'-O-Me modified nucleotides at US1 to US12;

[0466] c. 2'-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12;

[0467] d. 2'-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9 and H1-11;

[0468] e. 2'-F modified nucleotides between hairpin 1 and hairpin 2;

[0469] f. 2'-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14;

[0470] g. 2'-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13 and H2-15;

[0471] h. 2'-F modified nucleotides at the second to last and fourth to last nucleotides at the 3' end; and

[0472] i. 2'-O-Me modified nucleotides at the third to last and last nucleotides at the 3' end.

[0473] Embodiment 47. The sgRNA of Embodiment 46, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0474] Embodiment 48. A single guide RNA (sgRNA) comprising

[0475] a. 2'-O-Me modified nucleotides at LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15; and

[0476] b. 2'-F modified nucleotides at LS7, LS9, LS11; H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12 and H2-14.

[0477] Embodiment 49. The sgRNA of Embodiment 48, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0478] Embodiment 50. The sgRNA of any one of embodiments 48 to 49, further comprising

[0479] a. 2'-O-Me modified nucleotides at the last and third to last nucleotides at the 3' terminus; and

[0480] b. 2'-F modified nucleotides at the second to last and third to last nucleotides at the 3' end.

[0481] Embodiment 51. An sgRNA comprising a nucleic acid of any one of SEQ ID Nos: 228 to 332.

[0482] Embodiment 52. An sgRNA comprising a nucleic acid of any one of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329.

[0483] Embodiment 53. An sgRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity to the nucleic acid of any one of SEQ ID Nos: 235 to 240, 265 to 285 and 309 to 329, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier.

[0484] Embodiment 54. The sgRNA of any one of embodiments 51 to 53, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0485] B. Composition of dgRNA

[0486] In some embodiments, the compositions and methods of the present invention include gRNA, which includes crRNA and trRNA that guide a nuclease such as Cas9 to a target DNA sequence. In some embodiments, gRNA is combined on two independent RNA molecules (double guide RNA or dgRNA).

[0487] Table 2 and Fig. 21C A description of the domains of crRNA as used herein is provided. The 5' terminal region may include a spacer at or near the 5' end of the crRNA and be used to guide Cas9 to the target region domain in the DNA, for example as described herein. In Table 2, "n" between regions represents a variable number of nucleotides, such as 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some embodiments, n is equal to 0. Any of the dgRNAs described herein may include "n" between any domains.

[0488] Table 3 and Fig. 21C A description of the domains of trRNA as used herein is provided. In Table 3, "n" between regions represents a variable number of nucleotides, such as 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some embodiments, n is equal to 0. Any of the dgRNAs described herein may include "n" between any domains.

[0489] 1. Domain structure of dgRNA

[0490] As described in Briner 2014, dgRNAs can be developed based on specific functional domains referred to herein as "domains", including a spacer domain responsible for orientation, a lower stem, a protrusion, an upper stem, a linker, and a hairpin domain. In dgRNAs, crRNA contains some components of gRNA and trRNA contains some components of gRNA.

[0491] The regions of crRNA are provided in Table 2 and Fig. 21C The regions of trRNA are provided in Table 3 and Fig. 21C middle. Fig. 21C Schematic diagram showing exemplary dgRNAs.

[0492] Table 2: Regions of crRNA (linear view, 5′ to 3′)

[0493] LS1-6 B1-2 US1-14 5′ terminal region (n) lower stem area n Carina n upper stem area 3′ terminal region

[0494] Table 3: Regions of trRNA (linear view, 5′ to 3′)

[0495] US1-11 B1-4 LS1-6 N1-18 H1-1 to H1-12 H2-1 to H2-15 5' terminal region (n) upper stem area n Carina n lower stem area n Connection Area n Hairpin Zone 1 n Hairpin Zone 2 3′ terminal region

[0496] a) 5' terminal region

[0497] In some embodiments, the dgRNA comprises as shown in Tables 2-3 and Fig. 21C The nucleotides at the 5' end of the crRNA and trRNA shown in .

[0498] In some embodiments, the 5' end of the crRNA comprises a spacer or guide region for guiding the Cas protein to the target nucleotide sequence. In some embodiments, the 5' end does not comprise a spacer or guide region. In some embodiments, the 5' end comprises a spacer and additional nucleotides that are not used to guide the Cas protein to the target nucleotide region.

[0499] In some embodiments, the guide region comprises 1 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides before the 5' end of the crRNA. In some embodiments, the guide region comprises 20 nucleotides. In some embodiments, the guide region may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more than 25 nucleotides. In some embodiments, the guide region may comprise 17 nucleotides. In some embodiments, the guide region may comprise 18 nucleotides. In some embodiments, the guide region may comprise 19 nucleotides.

[0500] In some embodiments, the selection of the guide region is determined based on the target sequence within the gene of interest for editing. For example, in some embodiments, the crRNA comprises a guide region that is complementary to the target sequence of the gene of interest.

[0501] In some embodiments, the target sequence in the gene of interest may be complementary to the guide region of the crRNA. In some embodiments, the degree of complementarity or identity between the guide region of the crRNA and its corresponding target sequence in the gene of interest may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the guide region of the crRNA and the target region domain of the gene of interest may be 100% complementary or consistent. In other embodiments, the guide region of the crRNA and the target region domain of the gene of interest may contain at least one mismatch. For example, the guide region of the crRNA and the target sequence of the gene of interest may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches, wherein the total length of the target sequence is at least about 17, 18, 19, 20 or more than 20 base pairs. In some embodiments, the guide region of the crRNA and the target region domain of the target gene may contain 1 to 6 mismatches, wherein the guide sequence comprises at least about 17, 18, 19, 20 or more than 20 nucleotides. In some embodiments, the guide region of the crRNA and the target region domain of the target gene may contain 1, 2, 3, 4, 5 or 6 mismatches, wherein the guide sequence comprises about 20 nucleotides.

[0502] In some embodiments, the trRNA comprises a 5' terminal region. In some embodiments, the trRNA comprises a 5' terminal region that partially forms the upper stem region of the dgRNA. The 5' terminal region of the trRNA is not complementary to the region of the target gene.

[0503] b) Lower stem area

[0504] In some embodiments, the dgRNA comprises a lower stem (LS) region. The lower stem region comprises Fig. 21C In some embodiments, the lower stem region of crRNA and the lower stem region of trRNA are at least partially complementary to each other. In some embodiments, the lower stem region of crRNA and the lower stem region of trRNA are fully complementary to each other.

[0505] In some embodiments, the lower stem regions of crRNA and trRNA each comprise 6 nucleotides. In some embodiments, the lower stem regions of crRNA and trRNA each comprise 6 nucleotides. Fig. 21C In some embodiments, the lower stem region comprises fewer nucleotides than those shown in Tables 2-3 and Fig. 21C When the lower stem region contains more nucleotides than those shown in Tables 2-3 and Fig. 21CWhen less or more nucleotides are shown in the schematic diagram of, a modification pattern obvious to those skilled in the art is maintained. In some embodiments, the number of nucleotides in the lower stem region of crRNA is different from the number of nucleotides in the lower stem region of trRNA.

[0506] c) Protrusion area

[0507] In some embodiments, the dgRNA comprises a protrusion (B) region. In some embodiments, the crRNA comprises a protrusion region and the trRNA comprises a protrusion region. In some embodiments, each protrusion region comprises 1 to 4 nucleotides. In some embodiments, the protrusion region of the crRNA comprises two nucleotides, and the protrusion region of the trRNA comprises four nucleotides.

[0508] In some embodiments, the crRNA protrusion region is located between the lower stem region and the upper stem region of the crRNA. In some embodiments, the protrusion region of the crRNA comprises two nucleotides. In some embodiments, the protrusion region of the crRNA comprises as shown in Table 2 and Fig. 21C Nucleotides B1 and B2 are shown.

[0509] In some embodiments, the trRNA protuberance region is located between the upper stem region and the lower stem region of the trRNA. In some embodiments, the protuberance region of the trRNA comprises four nucleotides. In some embodiments, the protuberance region of the trRNA comprises as shown in Table 3 and Fig. 21C Nucleotides B1 to B4 are shown.

[0510] In some embodiments, the presence of the protuberance results in a directional kink between the upper and lower stem modules in the dgRNA. The crRNA protuberance and the trRNA protuberance may be partially complementary. The crRNA protuberance and the trRNA protuberance may not be complementary.

[0511] In some embodiments, the protuberance regions of crRNA and trRNA include the ratios shown in Tables 2-3 and Fig. 21C When the bulge region contains more nucleotides than those shown in Table 2-3 and Fig. 21C When fewer or more nucleotides are shown in the schematic diagram of , a modification pattern that is obvious to those skilled in the art is maintained. In some embodiments, the number of nucleotides in the protuberance region of the crRNA is different from the number of nucleotides in the protuberance region of the trRNA.

[0512] d) Upper stem region

[0513] In some embodiments, the dgRNA comprises an upper stem (US) region. The upper stem region comprises Fig. 21CIn some embodiments, the upper stem region of crRNA and the upper stem region of trRNA are at least partially complementary to each other. In some embodiments, the upper stem region of crRNA and the upper stem region of trRNA are fully complementary to each other.

[0514] In some embodiments, the upper stem region of the crRNA comprises fourteen nucleotides. In some embodiments, the upper stem region of the trRNA comprises eleven nucleotides. In some embodiments, the upper stem region of the crRNA and trRNA each comprises a ratio of Tables 2-3 and Fig. 21C In some embodiments, the upper stem region of crRNA and trRNA comprises less nucleotides than those shown in Tables 2-3 and Fig. 21C When the upper stem region contains more nucleotides than those shown in Table 2-3 and Fig. 21C When fewer or more nucleotides are shown in the schematic diagram, the modification pattern is maintained which is obvious to those skilled in the art.

[0515] In some embodiments, the upper stem of the crRNA comprises as shown in Table 2 and Fig. 21C Nucleotides US1 to US14 are shown in .

[0516] In some embodiments, the upper stem of trRNA comprises as shown in Table 3 and Fig. 21C Nucleotides US1 to US11 are shown in FIG.

[0517] e) Connection area

[0518] In some embodiments, the dgRNA comprises a trRNA comprising a connecting region. In some embodiments, the connecting region is between the lower stem region of the trRNA and the hairpin 1 region. In some embodiments, the connecting region is immediately downstream of the lower stem region of the trRNA. In some embodiments, the connecting region comprises eighteen nucleotides. In some embodiments, the connecting region of the trRNA comprises as shown in Table 3 and Fig. 21C In some embodiments, the linking region comprises nucleotides N1 to N18 as shown in Table 3 and Fig. 21C In some embodiments, the connecting region of the trRNA comprises less nucleotides than those shown in Table 3 and Fig. 21C When the linker region contains more than 3 and Fig. 21C When fewer or more nucleotides are shown, the modification pattern is maintained which is obvious to those skilled in the art.

[0519] In some embodiments, the connecting region has nucleotides that are complementary to the nucleic acid sequence when read in the opposite direction. In some embodiments, the complementarity in the nucleic acid sequence generates a secondary structure of the stem and / or stem loop in the sgRNA (e.g., certain nucleotides in the connecting region may base pair with each other). In some embodiments, the connecting regions may not be completely complementary to each other when read in the opposite direction.

[0520] f) Hairpin area

[0521] In some embodiments, the hairpin region of trRNA is downstream of the connecting region. In some embodiments, the region of nucleotides immediately downstream of the connecting region is referred to as "hairpin 1 region". In some embodiments, the region of nucleotides immediately downstream of the hairpin 1 region is referred to as "hairpin 2 region". In some embodiments, the hairpin region comprises hairpin 1 region and hairpin 2 region. In some cases, hairpin 1 region and hairpin 2 region are separated by one or more nucleotides "n". In some embodiments, n=1. In some embodiments, trRNA comprises only hairpin 1 region or hairpin 2 region.

[0522] It has been shown that replacing the hairpin 1 region of trRNA by 2 nucleotides allows the activity of editing Cas RNP (see US20150376586, Figure 16). In some embodiments, the trRNA comprises replacing the hairpin 1 region by nucleotide "n", wherein "n" is an integer between 1 and 50, 40, 30, 20, 15, 10, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of trRNA is replaced by 2 nucleotides.

[0523] In some embodiments, the hairpin 1 region of the trRNA comprises twelve nucleotides immediately downstream of the connecting region. In some embodiments, the hairpin 1 region of the trRNA comprises as shown in Table 3 and Fig. 21C Nucleotides H1-1 to H1-12 are shown in FIG.

[0524] In some embodiments, non-hairpin nucleotides are present between the hairpin 1 region and the hairpin 2 region of the trRNA. In some embodiments, one to two non-hairpin nucleotides are present between the hairpin 1 region and the hairpin 2 region.

[0525] In some embodiments, the hairpin 2 region of the trRNA comprises fifteen nucleotides after (3' to) hairpin 1. In some embodiments, the hairpin 2 region of the trRNA comprises as shown in Table 3 and Fig. 21C In some embodiments, the hairpin 2 region of the trRNA comprises nucleotides H2-1 to H2-15 as shown in Table 3, and "n" between the hairpin 1 region and the hairpin 2 region is 1 or 2.

[0526] In some embodiments, the hairpin region of the trRNA comprises a ratio of Table 3 and Fig. 21C When the hairpin region contains more nucleotides than those shown in Table 3 and Fig. 21C When fewer or more nucleotides are shown, the modification pattern is maintained which is obvious to those skilled in the art.

[0527] In some embodiments, when read in the opposite direction, the hairpin region has nucleotides that are complementary to the nucleic acid sequence. In some embodiments, when read in the opposite direction, the hairpin region may not be completely complementary to each other (e.g., the top or loop of the hairpin region contains unpaired nucleotides).

[0528] In some embodiments, the trRNA comprises a hairpin 1 region substituted with nucleotides "n", wherein "n" is an integer between 1 and 50, 40, 30, 20, 15, 10, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of the trRNA is substituted with 2 nucleotides.

[0529] g) 3' terminal region

[0530] In some embodiments, the dgRNA comprises a trRNA containing a 3' terminal region, and the 3' terminal region comprises additional nucleotides after the hairpin region (relative to it being 3'). In some embodiments, the 3' terminal region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more than 20 nucleotides that are not bound to the secondary structure of the hairpin region. In some embodiments, the 3' terminal region comprises 1, 2, 3 or 4 nucleotides that are not bound to the secondary structure of the hairpin region. In some embodiments, the 3' terminal region comprises 4 nucleotides that are not bound to the secondary structure of the hairpin region. In some embodiments, the 3' terminal region comprises 1, 2 or 3 nucleotides that are not bound to the secondary structure of the hairpin region.

[0531] 2. Modification of dgRNA

[0532] In some embodiments, the dgRNA comprises a modified crRNA and an unmodified trRNA. In some embodiments, the dgRNA comprises an unmodified crRNA and a modified trRNA. In some embodiments, the crRNA and trRNA of the dgRNA both comprise modifications.

[0533] In some embodiments, the gRNA described herein is on two independent RNA molecules (dual guide RNA or dgRNA). See Tables 2, 3 and Fig. 21C .

[0534] In some embodiments, the present invention comprises a dgRNA comprising or consisting of: a) any one of the crRNA sequences of SEQ ID Nos: 1 to 187; and b) any one of the trRNA sequences described in SEQ ID Nos: 188 to 227.

[0535] In some embodiments, a dgRNA comprising any one of the modified crRNA sequences 1 to 187 is provided.

[0536] In some embodiments, a dgRNA comprising any one of the modified trRNA sequences from 188 to 227 is provided.

[0537] In some embodiments, a dgRNA is provided comprising any one of the modified crRNA sequences of SEQ ID Nos: 19 to 31, 53 to 73, and 104 to 130. In some embodiments, the present invention comprises a dgRNA comprising any one of the modified sequences of SEQ ID Nos: 19 to 31, 53 to 73, and 104 to 130, wherein the crRNA further comprises a 5' spacer sequence that is at least partially complementary to the target sequence and guides Cas9 to its target for cleavage.

[0538] In some embodiments, the present invention comprises a crRNA comprising any one of the sequences described in SEQ ID Nos: 1 to 187. In some embodiments, the present invention comprises a crRNA comprising or consisting of any one of the sequences described in SEQ ID Nos: 19 to 31, 53 to 73, and 104 to 130. In some embodiments, the present invention comprises a crRNA comprising any one of the sequences described in SEQ ID Nos: 19 to 31, 53 to 73, and 104 to 130 and a spacer.

[0539] In some embodiments, the present invention comprises a trRNA comprising or consisting of any one of the sequences set forth in SEQ ID Nos: 188 to 277.

[0540] In some embodiments, the present invention comprises a crRNA comprising nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity with the nucleotides of any one of SEQ ID Nos: 1 to 187, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier. That is, the nucleotides A, U, C and G may differ by 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% compared to those shown in the sequence, but the modification remains the same.

[0541] In some embodiments, the invention comprises a trRNA comprising nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity to the nucleotides of any one of SEQ ID Nos: 188 to 277, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier. That is, the nucleotides A, U, C and G may differ by 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% compared to those shown in the sequence, but the modification on each nucleotide remains unchanged.

[0542] 3. Modified crRNA, trRNA and dgRNA

[0543] In some embodiments, the crRNA comprises one or more modified nucleotides within one or more of the 5' terminal region, the lower stem region, the protuberance region, the upper stem region, and the 3' terminal region.

[0544] In some embodiments, the modification comprises 2'-O-Me.

[0545] In some embodiments, the modification comprises a 2'-F.

[0546] In some embodiments, the modification comprises a phosphorothioate (PS) bond linking one or more nucleotides. In some embodiments, the modification is three PS bonds linking the first four nucleotides at the 5' end and three PS bonds linking the last four nucleotides at the 3' end.

[0547] In some embodiments, the modification comprises an inverted abasic nucleotide.

[0548] In some embodiments, a crRNA is provided that comprises a 2'-O-Me modified nucleotide at each nucleotide in the upper stem region. In some embodiments, US-1 to US-14 of the crRNA are each modified with 2'-O-Me. In some embodiments, LS1 and LS6 of the crRNA are modified with 2'-O-Me. In some embodiments, LS5 of the crRNA is modified with 2'-O-Me.

[0549] In some embodiments, a crRNA is provided, comprising each of the nucleotides in the upper stem region, and 2'-O-Me modified nucleotides at LS1 and LS6 in the lower stem region. In some embodiments, the crRNA further comprises one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as 5' and / or 3' terminal modifications.

[0550] In some embodiments, a crRNA is provided, comprising each of the nucleotides in the upper stem region, 2'-O-Me modified nucleotides at LS1, LS5, and LS6 in the lower stem region. In some embodiments, the crRNA further comprises one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as 5' and / or 3' terminal modifications.

[0551] In some embodiments, the present invention comprises a crRNA comprising 2'-F modified nucleotides at LS1, LS2 and LS6 in the lower stem region. In some embodiments, the crRNA further comprises 2'-F modified nucleotides at each of B1 and B2 in the protrusion region. In some embodiments, the present invention comprises a crRNA comprising LS1, LS2 and LS6 in the lower stem region, and 2'-F modified nucleotides at each of B1 and B2 in the protrusion region. In some embodiments, the crRNA further comprises one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as 5' and / or 3' end modifications.

[0552] In some embodiments, the crRNA comprises nucleotides LS1 and LS6 in the lower stem region; each of the nucleic acids in the protrusion region; and 2'-O-Me modified nucleotides at each of the nucleic acids in the upper stem region. In some embodiments, the LS5 nucleotide of the crRNA is also modified with 2'-O-Me. In some embodiments, LS2, LS3, and LS4 of the crRNA are unmodified. In some embodiments, the crRNA further comprises one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as 5' and / or 3' end modifications.

[0553] In some embodiments, crRNA comprises LS1, LS2 and LS6 in the lower stem region, and nucleotides in the projection region at each 2'-fluoro (2'-F) modified nucleotides. In some embodiments, crRNA comprises LS1, LS2 and LS6 in the lower stem region, and nucleotides in the projection region at each 2'-fluoro (2'-F) modified nucleotides. In some embodiments, crRNA comprises LS1, LS2 and LS6 in the lower stem region, and nucleotides in the projection region at each 2'-fluoro (2'-F) modified nucleotides. In some embodiments, crRNA comprises LS1-LS6 in the lower stem region, and nucleotides in the projection region at each 2'-fluoro (2'-F) modified nucleotides. In some embodiments, crRNA further comprises 5' and / or 3' terminal regions, such as 5' and / or 3' end modifications in one or more 2'-O-Me or 2'-O-moe modified nucleotides.

[0554] In some embodiments, the invention comprises a trRNA comprising one or more modified nucleotides within one or more of the following regions: a 5' terminal region; an upper stem region; a protuberance region; a lower stem region; a connecting region; a hairpin 1 region; an interventing region between the hairpin 1 region and the hairpin 2 region; a hairpin 2 region; and a 3' terminal region.

[0555] In some embodiments, the modification comprises 2'-O-Me.

[0556] In some embodiments, the modification comprises a 2'-F.

[0557] In some embodiments, the modification comprises a phosphorothioate (PS) bond linking one or more nucleotides. In some embodiments, the modification is three PS bonds linking the first four nucleotides at the 5' end and three PS bonds linking the last four nucleotides at the 3' end.

[0558] In some embodiments, the modification comprises an inverted abasic nucleotide.

[0559] In some embodiments, the trRNA comprises 2'-O-Me modified nucleotides at each of the following: each nucleic acid in the upper stem region; B1 and B2 in the protuberance region; LS1 and LS2 in the lower stem region; N3, N4, N5, N15, N16, N17, and N18 in the connecting region; each nucleotide in the hairpin 1 region; one nucleotide between the hairpin 1 region and the hairpin 2 region; and each nucleotide in the hairpin 2 region. In some embodiments, the trRNA further comprises one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as in the 5' and / or 3' terminal modifications.

[0560] In some embodiments, the trRNA comprises 2'-O-Me modified nucleotides at each of the following: each nucleic acid in the upper stem region; each nucleotide in the protrusion region; LS1, LS2, LS5, and LS6 in the lower stem region; N3 to N5, N10 to N18 in the connecting region; each nucleotide in the hairpin 1 region; one nucleotide between the hairpin 1 region and the hairpin 2 region; and each nucleotide in the hairpin 2 region. In some embodiments, the crRNA further comprises one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as in the 5' and / or 3' terminal modifications.

[0561] In some embodiments, the trRNA comprises 2'-F modified nucleotides at N15 to N18 in the linker region. In some embodiments, the trRNA further comprises one or more 2'-F modified nucleotides in the 5' and / or 3' terminal regions, e.g., in the 5' and / or 3' terminal modifications.

[0562] In some embodiments, the trRNA comprises LS4 and LS5 in the lower stem region, and 2'-F modified nucleotides at N13 to N18 in the connecting region. In some embodiments, the trRNA further comprises one or more 2'-F modified nucleotides in the 5' and / or 3' terminal regions, e.g., in the 5' and / or 3' terminal modifications.

[0563] In some embodiments, the trRNA comprises 2'-F modified nucleotides at LS1, LS3, and LS5 in the lower stem region, and 2'-O-Me modified nucleotides at LS2, LS4, and LS6 in the lower stem region.

[0564] In some embodiments, disclosed herein is a CRISPR RNA (crRNA) comprising one or more modifications in one or more of the following regions: the first five nucleotides at the 5' end; the lower stem region; the protrusion region; the upper stem region; and the last five nucleotides at the 3' end. In some embodiments, the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the modification comprises a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end are modified. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a phosphorothioate (PS) bond. In some embodiments, the modification comprises 2'-O-Me. In some embodiments, the modification comprises 2'-F. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a PS bond, and wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2'-O-Me modification. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a PS bond, and wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2'-F modification. In some embodiments, LS1 and LS6 are modified with 2'-O-Me. In some embodiments, each of the nucleotides in the upper stem region is modified with 2'-O-Me.

[0565] In some embodiments, the present invention comprises a crispr RNA (crRNA) comprising a 2'-O-Me modified nucleic acid at the following nucleotides: LS1 and LS6 in the lower stem region; and each nucleotide in the upper stem region. In some embodiments, the crRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the crRNA further comprises a 2'-O-Me or 2'-F modified nucleic acid at the first three nucleotides at the 5' end, and a 2'-O-Me or 2'-F modified nucleic acid at the last three nucleotides at the 3' end. In some embodiments, LS1, LS2, and LS6 are modified with 2'-F. In some embodiments, each nucleotide in the protrusion region is modified with 2'-F.

[0566] In some embodiments, disclosed herein is a crispr RNA (crRNA), comprising a 2'-F modified nucleic acid at the following nucleotides: LS1, LS2, and LS6 in the lower stem region; and each nucleotide in the protrusion region. In some embodiments, the crRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the crRNA further comprises a 2'-O-Me or 2'-F modified nucleic acid at the first three nucleotides at the 5' end, and a 2'-O-Me or 2'-F modified nucleic acid at the last three nucleotides at the 3' end.

[0567] In some embodiments, a crRNA is provided that comprises a nucleic acid of any one of SEQ ID Nos: 1 to 187. In some embodiments, a crRNA is provided that comprises a nucleic acid of any one of SEQ ID Nos: 19 to 31, 53 to 73, 104 to 130, and 161 to 187. In some embodiments, a crRNA is provided that comprises a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to a nucleic acid of any one of SEQ ID Nos: 19 to 31, 53 to 73, 104 to 130, and 161 to 187, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier. In some embodiments, the crRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0568] The present invention also encompasses tracr RNA (trRNA) comprising one or more modifications in one or more of the following regions: the first five nucleotides at the 5' end; the upper stem region; the protrusion region; the lower stem region; the connecting region; the hairpin 1 region; the hairpin 2 region; and the last five nucleotides at the 3' end. In some embodiments, the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the modification comprises a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are connected to a phosphorothioate (PS) bond. In some embodiments, the first three nucleotides at the 5' end and the last three nucleotides at the 3' end are modified. In some embodiments, the modification comprises 2'-O-Me. In some embodiments, the modification comprises 2'-F. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are connected to the PS key, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end are modified with 2'-O-Me. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are connected to the PS key, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end are modified with 2'-F. In some embodiments, each nucleotide in the upper stem region is modified with 2'-O-Me. In some embodiments, B1 and B2 in the protrusion region are modified with 2'-O-Me. In some embodiments, N3, N4, N5, N15, N16, N17 and N18 in the connection region are modified with 2'-O-Me. In some embodiments, each nucleotide in the hairpin 1 region is modified with 2'-O-Me. In some embodiments, each nucleotide in the hairpin 2 region is modified with 2'-O-Me.

[0569] In some embodiments, the present invention comprises tracrRNA (trRNA), which comprises 2'-O-Me modified nucleic acids at the following nucleotides: each nucleotide in the upper stem region; B1 and B2 in the protrusion region; N3, N4, N5, N15, N16, N17 and N18 in the connecting region; each nucleotide in the hairpin 1 region; and each nucleotide in the hairpin 2 region. In some embodiments, trRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, trRNA further comprises 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end, and 2'-O-Me or 2'-F modified nucleic acids at the last three nucleotides at the 3' end. In some embodiments, N15, N16, N17 and N18 are modified with 2'-F. In some embodiments, LS1, LS3, and LS5 are modified with 2'-F, and LS2, LS4, and LS6 are modified with 2'-O-Me. In some embodiments, the trRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the trRNA further comprises a 2'-O-Me or 2'-F modified nucleic acid at the first three nucleotides at the 5' end, and a 2'-O-Me or 2'-F modified nucleic acid at the last three nucleotides at the 3' end.

[0570] In some embodiments, a trRNA is provided comprising a nucleic acid of any one of SEQ ID Nos: 188 to 227. In some embodiments, a trRNA is provided comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to a nucleic acid of any one of SEQ ID Nos: 188 to 227, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier. In some embodiments, the trRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0571] In some cases, a dual guide comprising a crRNA and a trRNA is provided, wherein the crRNA comprises a nucleic acid of any one of SEQ ID Nos: 1-187, and wherein the trRNA comprises a nucleic acid of any one of SEQ ID Nos: 188-227.

[0572] The present invention encompasses dual guides comprising a crRNA disclosed herein and a trRNA disclosed herein, as well as dual guides comprising a crRNA disclosed herein and an unmodified trRNA. In some embodiments, a dual guide comprising an unmodified crRNA and a modified trRNA disclosed herein is provided.

[0573] In some embodiments, the following are contemplated:

[0574] Embodiment 55. A crisprRNA (crRNA) comprising one or more modifications in one or more of the following regions:

[0575] a. The first five nucleotides at the 5' end;

[0576] b. Lower stem region;

[0577] c. protrusion area;

[0578] d. upper stem region; and

[0579] e. The last five nucleotides at the 3' end.

[0580] Embodiment 56. The crRNA of Embodiment 55, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0581] Embodiment 57. The crRNA of Embodiment 55, wherein the modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[0582] Embodiment 58. The crRNA of Embodiment 55, wherein the modification comprises a phosphorothioate (PS) bond between nucleotides.

[0583] Embodiment 59. The crRNA of any one of Embodiments 55 to 58, wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end are modified.

[0584] Embodiment 60. The crRNA of any one of embodiments 55 to 58, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to phosphorothioate (PS) bonds.

[0585] Embodiment 61. The crRNA of Embodiment 59, wherein the modification comprises 2'-O-Me.

[0586] Embodiment 62. The crRNA of Embodiment 59, wherein the modification comprises 2'-F.

[0587] Embodiment 63. The crRNA of any one of Embodiments 55 to 62, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a PS bond, and wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2'-O-Me modification.

[0588] Embodiment 64. The crRNA of any one of Embodiments 55 to 62, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to a PS bond, and wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise a 2'-F modification.

[0589] Embodiment 65. The crRNA of any one of Embodiments 55 to 60, wherein LS1 and LS6 are modified with 2'-O-Me.

[0590] Embodiment 66. The crRNA of any one of embodiments 55 to 60 and 65, wherein each of the nucleotides in the upper stem region is modified with 2'-O-Me.

[0591] Embodiment 67. A crispr RNA (crRNA) comprising a 2'-O-Me modified nucleotide at:

[0592] a. LS1 and LS6 in the lower stem region; and

[0593] b. Each nucleotide in the upper stem region.

[0594] Embodiment 68. The crRNA of Embodiment 67, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0595] Embodiment 69. The crRNA of embodiment 67 or 68, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end, and 2'-O-Me or 2'-F modified nucleotides at the last three nucleotides at the 3' end.

[0596] Embodiment 70. The crRNA of any one of Embodiments 55 to 60, wherein LS1, LS2 and LS6 are modified with 2'-F.

[0597] Embodiment 71. The crRNA of any one of Embodiments 55 to 60 and 70, wherein each nucleotide in the protuberance region is modified with 2'-F.

[0598] Embodiment 72. A crispr RNA (crRNA) comprising a 2'-F modified nucleotide at:

[0599] a. LS1, LS2 and LS6 in the lower stem region; and

[0600] b. Each nucleotide in the bulge region.

[0601] Embodiment 73. The crRNA of any one of Embodiments 70 to 72, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0602] Embodiment 74. The crRNA of embodiment 72 or 73, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end, and 2'-O-Me or 2'-F modified nucleotides at the last three nucleotides at the 3' end.

[0603] Embodiment 75. A crRNA comprising a nucleic acid of any one of SEQ ID Nos: 1 to 187.

[0604] Embodiment 76. A crRNA comprising a nucleic acid of any one of SEQ ID Nos: 19 to 31, 53 to 73, 104 to 130, and 161 to 187.

[0605] Embodiment 77. A crRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity to the nucleic acid of any one of SEQ ID Nos: 19 to 31, 53 to 73, 104 to 130 and 161 to 187, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier.

[0606] Embodiment 78. The crRNA of any one of Embodiments 75 to 77, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0607] Embodiment 79. A tracrRNA (trRNA) comprising one or more modifications in one or more of the following regions:

[0608] a. The first five nucleotides at the 5' end;

[0609] b. Upper stem region;

[0610] c. protrusion area;

[0611] d. Lower stem region;

[0612] e. Connection area;

[0613] f. Hairpin 1 area;

[0614] g. Hairpin 2 area; and

[0615] h. Last five nucleotides at the 3' end.

[0616] Embodiment 80. The trRNA of embodiment 79, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0617] Embodiment 81. The trRNA of Embodiment 79, wherein the modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[0618] Embodiment 82. The trRNA of embodiment 79, wherein the modification comprises a phosphorothioate (PS) bond between nucleotides.

[0619] Embodiment 83. The trRNA of any one of embodiments 79 to 82, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked to phosphorothioate (PS) bonds.

[0620] Embodiment 84. The trRNA of any one of embodiments 79 to 82, wherein the first three nucleotides at the 5' end and the last three nucleotides at the 3' end are modified.

[0621] Embodiment 85. The trRNA of Embodiment 84, wherein the modification comprises 2'-O-Me.

[0622] Embodiment 86. The trRNA of Embodiment 84, wherein the modification comprises 2'-F.

[0623] Embodiment 87. The trRNA of any one of embodiments 79 to 86, wherein the first four nucleotides at the 5' terminus and the last four nucleotides at the 3' terminus are linked to a PS bond, and wherein the first three nucleotides at the 5' terminus and the last three nucleotides at the 3' terminus comprise a 2'-O-Me modification.

[0624] Embodiment 88. The trRNA of any one of embodiments 79 to 86, wherein the first four nucleotides at the 5' terminus and the last four nucleotides at the 3' terminus are linked to a PS bond, and wherein the first three nucleotides at the 5' terminus and the last three nucleotides at the 3' terminus comprise a 2'-F modification.

[0625] Embodiment 89. The trRNA of any one of embodiments 79 to 84, wherein each nucleotide in the upper stem region is modified with 2'-O-Me.

[0626] Embodiment 90. The trRNA of any one of embodiments 79 to 84 and 89, wherein B1 and B2 within the protuberance region are modified with 2'-O-Me.

[0627] Embodiment 91. The trRNA of any one of embodiments 79 to 84 and 89 to 90, wherein N3, N4, N5, N15, N16, N17 and N18 in the linker region are modified with 2'-O-Me.

[0628] Embodiment 92. The trRNA of any one of embodiments 79 to 84 and 89 to 91, wherein each nucleotide in the hairpin 1 region is modified with 2'-O-Me.

[0629] Embodiment 93. The trRNA of any one of embodiments 79 to 84 and 89 to 92, wherein each nucleotide in the hairpin 2 region is modified with 2'-O-Me.

[0630] Embodiment 94. A tracrRNA (trRNA) comprising 2'-O-Me modified nucleotides at:

[0631] a. Each nucleotide in the upper stem region;

[0632] b. B1 and B2 in the protrusion area;

[0633] c. N3, N4, N5, N15, N16, N17 and N18 in the connection area;

[0634] d. each nucleotide in the hairpin 1 region; and

[0635] e. Each nucleotide in the hairpin 2 region.

[0636] Embodiment 95. The trRNA of Embodiment 94, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0637] Embodiment 96. The crRNA of embodiment 94 or 95, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end, and 2'-O-Me or 2'-F modified nucleic acids at the last three nucleotides at the 3' end.

[0638] Embodiment 97. The trRNA of any one of embodiments 79 to 84, wherein N15, N16, N17 and N18 are modified with 2'-F.

[0639] Embodiment 98. The trRNA of any one of embodiments 79 to 84 and 97, wherein LS1, LS3 and LS5 are modified with 2'-F, and LS2, LS4 and LS6 are modified with 2'-O-Me.

[0640] Embodiment 99. The trRNA of any one of embodiments 87 to 98, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0641] Embodiment 100. The trRNA of embodiment 98 or 99, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' terminus, and 2'-O-Me or 2'-F modified nucleotides at the last three nucleotides at the 3' terminus.

[0642] Embodiment 101. A trRNA comprising a nucleic acid of any one of SEQ ID Nos: 188 to 227.

[0643] Embodiment 102. A trRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity to the nucleic acid of any one of SEQ ID Nos: 188 to 227, wherein the modification pattern is the same as the modification pattern shown in the reference sequence identifier.

[0644] Embodiment 103. The trRNA of any one of embodiments 101 to 102, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0645] Embodiment 104. A dual guide comprising crRNA and trRNA, wherein the crRNA comprises the nucleotides of any one of SEQ ID Nos: 1 to 187, and wherein the trRNA comprises the nucleic acids of any one of SEQ ID Nos: 188 to 227.

[0646] Embodiment 105. A dual guide comprising the crRNA of any one of embodiments 55 to 78 and the trRNA of any one of embodiments 79 to 103.

[0647] Embodiment 106. A dual guide comprising the crRNA of any one of embodiments 55 to 78 and an unmodified trRNA.

[0648] Embodiment 107. A dual guide comprising an unmodified crRNA and the trRNA of any one of embodiments 79 to 103.

[0649] C. Modification of terminal nucleotides

[0650] In some embodiments, the 5' or 3' terminal nucleotide of any of the guide RNAs described herein is modified. In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region of the guide RNA, including, for example, sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, are modified. In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region of the guide RNA include more than one modification. In some embodiments, at least one of the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 3' terminal region is modified. In some embodiments, at least two of the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region are modified. In some embodiments, at least three of the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region are modified. In some embodiments, the modification comprises a PS bond.

[0651] In some embodiments, the 5' end of the 5' terminal region is modified, for example, sgRNA, dgRNA, crRNA, trRNA, or 1, 2, 3, 4, 5, 6, or 7 nucleotides before crRNA and trRNA are modified. In some embodiments, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region of the guide RNA include more than one modification. In some embodiments, at least one of the terminal (i.e., the first) 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5' end is modified. In some embodiments, at least two of the terminal 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5' end are modified. In some embodiments, at least three of the terminal 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5' end are modified. In some embodiments, the modification comprises a PS bond.

[0652] In some embodiments, the 5' and 3' ends (e.g., ends) of the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA are modified. In some embodiments, only the 5' end of the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA is modified. In some embodiments, only the 3' end of the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA is modified.

[0653] In some embodiments, the gRNA comprises a modification at 1, 2, 3, 4, 5, 6, or 7 of the first 7 nucleotides at the 5' end of the gRNA. In some embodiments, the gRNA comprises a modification at 1, 2, 3, 4, 5, 6, or 7 of the 7 terminal nucleotides at the 3' end. In some embodiments, 2, 3, or 4 of the first 4 nucleotides at the 5' end, and / or 2, 3, or 4 of the terminal 4 nucleotides at the 3' end are modified. In some embodiments, 2, 3, or 4 of the first 4 nucleotides at the 5' end are linked to phosphorothioate (PS) bonds.

[0654] In some embodiments, the modification to the 5' end and / or the 3' end comprises a 2'-O-methyl (2'-O-Me) or 2'-O-(2-methoxyethyl) (2'-O-moe) modification to the nucleotide. In some embodiments, the modification comprises a 2'-fluoro (2'-F) modification to the nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the modification comprises a reverse abasic nucleotide. In some embodiments, the modification comprises more than one modification selected from 2'O-Me, 2'-O-moe, 2'-fluoro (2'-F), a phosphorothioate (PS) bond between nucleotides, and a reverse abasic nucleotide. In some embodiments, equivalent modifications are contemplated.

[0655] In some embodiments, guide RNA, such as sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, include one or more thiophosphate (PS) bonds between the first, two, three, four, five, six, or seven nucleotides at the 5' end. In some embodiments, guide RNA, such as sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA include one or more PS bonds between the first, two, three, four, five, six, or seven nucleotides at the 3' end. In some embodiments, guide RNA, such as sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA include one or more PS bonds between the last, two, three, four, five, six, or seven nucleotides at the 5' end and the 3' end. In some embodiments, in addition to the PS bonds, the 5' and 3' terminal nucleotides may include 2'-O-Me, 2'-O-moe, or 2'-F modified nucleotides.

[0656] In some embodiments, the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, comprises modified nucleotides at the 5' and 3' ends, and Tables 1-3 and Fig.21Aor modified nucleotides in one or more other regions described in 21C.

[0657] In some embodiments, crRNA, trRNA, or both crRNA and trRNA comprise modified nucleotides that are not at the 5' or 3' end. Specific modification patterns are described below and in Table 4.

[0658] 3. Delivery of gRNA and Cas protein

[0659] In some embodiments, in addition to at least one gRNA, the compositions provided herein further include a nuclease. In some embodiments, the nuclease is a Cas protein. In some embodiments, gRNA together with the Cas protein is referred to as CasRNP. In some embodiments, the Cas protein is from a Type-II CRISPR / Cas system. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas9 protein is a wild-type Cas9. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes Cas9 protein, such as Streptococcus pyogenes Cas9. In some embodiments, the Cas9 protein is not derived from Streptococcus pyogenes, but works in the same manner as Streptococcus pyogenes Cas9, so that a gRNA specific for Streptococcus pyogenes Cas9 will guide non-Streptococcus pyogenes Cas9 to its target site. In some embodiments, Cas induces double-strand breaks in the target DNA. The embodiments described herein encompass equivalents of Streptococcus pyogenes Cas9 protein.

[0660] Cas9 encompasses modified forms and variants thereof. A modified form of Cas9 with an inactive catalytic domain (RuvC or HNH) is referred to as a "nickase". A nickase cuts only one strand on the target DNA, thus producing a single-strand break. A single-strand break may also be referred to as a "nick". In some embodiments, the compositions and methods include a nickase. In some embodiments, the compositions and methods include a nickase Cas9 that induces a nick rather than a double-strand break in the target DNA.

[0661] In some embodiments, the Cas protein can be modified to contain only one functional nuclease domain. For example, the Cas protein can be modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase Cas with a RuvC domain with reduced activity is used. In some embodiments, a nickase Cas with an inactive RuvC domain is used. In some embodiments, a nickase Cas with a HNH domain with reduced activity is used. In some embodiments, a nickase Cas with an inactive HNH domain is used.

[0662] In some embodiments, the conserved amino acids in the nuclease domain of the Cas protein are replaced to reduce or change the nuclease activity. In some embodiments, the Cas protein may include amino acid substitutions in RuvC or RuvC-like nuclease domains. Exemplary amino acid substitutions in RuvC or RuvC-like nuclease domains include D10A (based on Streptococcus pyogenes Cas9 protein). In some embodiments, the Cas protein may include amino acid substitutions in HNH or HNH-like nuclease domains. Exemplary amino acid substitutions in HNH or HNH-like nuclease domains include E762A, H840A, N863A, H983A, and D986A (based on Streptococcus pyogenes Cas9 protein).

[0663] In some embodiments, the RNP complex described herein comprises a nickase and a pair of guide RNAs complementary to the sense strand and antisense strand of the target sequence, respectively. In this embodiment, the guide RNA guides the nickase to the target sequence and introduces a double-strand break (DSB) by creating a nick (i.e., a double nick) on the relative strand of the target sequence. In some embodiments, the use of a double nick can improve specificity and reduce off-target effects. In some embodiments, two independent guide RNAs of the relative strands of the targeted DNA use a nickase Cas to create a double nick in the target DNA. In some embodiments, a nickase Cas is used together with two independent guide RNAs selected to be very close to each other to create a double nick in the target DNA.

[0664] In some embodiments, a chimeric Cas protein is used in which one domain or region of a protein is replaced with a portion of a different protein. In some embodiments, the Cas nuclease domain can be replaced with a domain from a different nuclease such as Fok1. In some embodiments, the Cas protein can be modified with a nuclease.

[0665] In some embodiments, the Cas protein comprises a fusion protein comprising a catalytically inactive Cas9 (see, e.g., WO2014152432) connected to a heterologous functional domain. In some embodiments, the catalytically inactive Cas9 is from Streptococcus pyogenes. In some embodiments, the catalytically inactive Cas9 comprises a mutation that inactivates 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.

[0666] A. PAM

[0667] In some embodiments, the target sequence may be adjacent to the 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 sequence of the PAM may depend on the Cas protein used. For example, the PAM can be selected from a consensus sequence or a specific PAM sequence for a specific Cas9 protein or Cas9 ortholog, including those disclosed in Ran et al., Nature 520:186-191 (2015). Figure 1 In those of, the document is incorporated herein by reference. In some embodiments, the PAM may include nucleotides of length 2, 3, 4, 5, 6, 7, 8, 9 or 10. 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 A or T, and R is defined as 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.

[0668] B. Delivery of Modified gRNA

[0669] Lipid nanoparticles (LNP) are a well-known method for delivering nucleotides and protein cargoes, and can be used to deliver gRNA, mRNA, Cas9 and RNP disclosed herein. In some embodiments, LNP delivers nucleic acids, proteins, or nucleic acids and proteins.

[0670] In some embodiments, the present invention comprises a method of delivering any one of the gRNAs disclosed herein to an individual, wherein the gRNA is combined with LNP. In some embodiments, the gRNA / LNP is also combined with Cas9 or mRNA encoding Cas9.

[0671] In some embodiments, the present invention comprises a composition containing any one of the disclosed gRNAs and LNP. In some embodiments, the composition further comprises Cas9 or an mRNA encoding Cas9.

[0672] In some embodiments, LNP comprises cationic lipid. In some embodiments, LNP comprises (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecanoate-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadecanoate-9,12-dienoate). In some embodiments, LNPs comprise a molar ratio (N:P) of cationic lipid amine to RNA phosphate of about 4.5.

[0673] In some embodiments, LNPs conjugated to a gRNA disclosed herein are used to prepare a medicament for treating a disease or disorder.

[0674] Electroporation is a well-known method for delivering cargo, and any electroporation method can be used to deliver any of the gRNAs disclosed herein. In some embodiments, electroporation can be used to deliver any of the gRNAs disclosed herein and Cas9 or mRNA encoding Cas9.

[0675] In some embodiments, the present invention comprises a method of delivering any one of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNA is bound to or not bound to LNP. In some embodiments, the gRNA / LNP or gRNA is also bound to Cas9 or mRNA encoding Cas9.

[0676] 4. Gene Regulation Methods

[0677] In some embodiments, the present invention comprises a pharmaceutical preparation comprising any of the gRNAs disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the present invention comprises a pharmaceutical preparation comprising any of the gRNAs disclosed herein and LNPs and a pharmaceutically acceptable carrier. In some embodiments, the present invention comprises a pharmaceutical preparation comprising any of the gRNAs disclosed herein, a Cas9 protein or an mRNA encoding a Cas9 protein, an LNP, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical preparation is used to prepare a drug for treating a disease or condition. In some embodiments, the present invention comprises a method for treating a human patient comprising administering any of the gRNAs or pharmaceutical preparations described herein.

[0678] In some embodiments, the present invention comprises a method or use of modifying a target DNA comprising administering or delivering a Cas protein or Cas mRNA and any one or more of the gRNAs disclosed herein.

[0679] In some embodiments, the present invention includes methods or uses for regulating a target gene, comprising administering or delivering any one or more of a Cas protein or a Cas mRNA and a gRNA disclosed herein. In some embodiments, regulation is target gene editing. In some embodiments, regulation is a change in the expression of a protein encoded by a target gene.

[0680] In some embodiments, the method or use results in gene editing. In some embodiments, the method or use results in double-strand breaks in the target gene. In some embodiments, the method or use results in the formation of insertion-deletion mutations during the non-homologous end joining of DSB. In some embodiments, the method or use results in the insertion or deletion of nucleotides in the target gene. In some embodiments, the insertion or deletion of nucleotides in the target gene results in a frameshift mutation or premature termination codon, which produces a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in the target gene results in knockout or elimination of target gene expression. In some embodiments, the method or use comprises homology-directed repair of DSB. In some embodiments, the method or use further comprises delivering a template to a cell, wherein at least a portion of the template is incorporated into a target DNA at or near a double-strand break site induced by a Cas protein.

[0681] In some embodiments, the method or use results in gene regulation. In some embodiments, gene regulation is an increase or decrease in gene expression, a change in the methylation state of DNA, or a modification of a histone subunit. In some embodiments, the method or use results in an increase or decrease in the expression of a protein encoded by a target gene.

[0682] In some embodiments, any of the gRNAs disclosed herein may be useful in the preparation of a medicament for treating a disease or disorder.

[0683] A. Measurement of Gene Regulation

[0684] The efficacy of the modified gRNA can be tested in vitro and in vivo. In some embodiments, the present invention comprises one or more of the gRNAs disclosed herein, wherein the gRNA results in gene regulation when provided to a cell together with Cas9. In some embodiments, the efficacy of the gRNA can be measured in an in vitro or in vivo assay.

[0685] 1. Measuring Cas efficacy in vitro

[0686] In some embodiments, the activity of a Cas RNP comprising a modified sgRNA is compared to the activity of a Cas RNP comprising an unmodified sgRNA.

[0687] In some embodiments, the activity of a Cas RNP comprising a dgRNA comprising a modified trRNA is compared to the activity of a Cas RNP comprising a dgRNA comprising an unmodified trRNA.

[0688] In some embodiments, the activity of a Cas RNP comprising a dgRNA comprising a modified crRNA is compared to the activity of a Cas RNP comprising a dgRNA comprising an unmodified crRNA.

[0689] In some embodiments, the activity of a Cas RNP comprising a dgRNA comprising a modified crRNA and a modified trRNA is compared to the activity of a Cas RNP comprising an unmodified crRNA and an unmodified trRNA.

[0690] In some embodiments, the efficiency of gRNA to increase or decrease target protein expression is determined by measuring the amount of target protein. In some embodiments, the present invention comprises any of the gRNAs described herein, wherein the gRNA increases or decreases the amount of protein produced from the targeted gene. In some embodiments, the present invention comprises a method of regulating protein expression, comprising administering any of the gRNAs disclosed herein to an individual, wherein the gRNA guides Cas9 to a gene encoding a target protein, and compared to a gRNA control that does not target Cas9 to the gene, the target protein expression is increased or decreased.

[0691] In some embodiments, the efficiency of editing by a specific gRNA is determined by editing at the target position in the genome after delivering Cas9 and gRNA (sgRNA or dgRNA comprising crRNA and trRNA). In some embodiments, the efficiency of editing by a specific gRNA is measured by second-generation sequencing. In some embodiments, the editing percentage of the target region domain is determined. In some embodiments, the total number of sequence reads with nucleotide insertions or deletions in the target region domain is measured compared to the total number of sequence reads after delivering gRNA and Cas9. In some embodiments, the present invention includes a method for increasing gene editing efficiency, comprising administering or delivering any of the modified gRNAs described herein to a cell, wherein the percentage of gene editing is increased compared to a control gRNA that has not been similarly modified.

[0692] In some embodiments, the efficiency of editing by a specific gRNA is measured by the presence of nucleotide insertions or deletions introduced by successful gene editing. In some embodiments, the present invention includes a method for producing nucleotide insertions or deletions in a gene, comprising administering or delivering any of the modified gRNAs described herein to a cell, wherein the nucleotides are inserted or deleted compared to a control gRNA that is not similarly modified. In some embodiments, the activity of Cas9 and gRNA is tested in a biochemical assay. In some embodiments, the activity of Cas9 and gRNA is tested in a cell-free cleavage assay. In some embodiments, the activity of Cas9 and gRNA is tested in Neuro2A cells.

[0693] In some embodiments, Cas9 and sgRNA or dgRNA comprising modified crRNA and / or trRNA show similar, greater, or reduced activity compared to unmodified sgRNA or dgRNA comprising unmodified crRNA and trRNA. In some embodiments, Cas9 and modified sgRNA or dgRNA comprising modified crRNA and / or trRNA show enhanced activity compared to unmodified sgRNA or dgRNA comprising unmodified crRNA and trRNA.

[0694] 2. Measuring Cas efficacy in vivo

[0695] In some embodiments, the activity of the modified gRNA is measured following in vivo administration of LNPs comprising the modified gRNA and a Cas protein or mRNA encoding a Cas protein.

[0696] In some embodiments, the in vivo efficacy of a gRNA or composition provided herein is determined by measuring the efficacy of editing DNA extracted from a tissue (e.g., liver tissue) following administration of the gRNA and Cas9.

[0697] 3. In vivo measurement of immune system activation

[0698] Modification of gRNA as disclosed herein can reduce the immune response of an individual to the administration of gRNA in vivo. In some embodiments, the activation of the immune response of an individual is measured by the serum concentration of one or more cytokines after in vivo administration of sgRNA or dgRNA comprising trRNA and crRNA together with Cas9 mRNA or protein (e.g., formulated in LNP). In some embodiments, the cytokine is interferon-α (IFN-α), interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1) and / or tumor necrosis factor α (TNF-α). In some embodiments, the present invention includes a method for reducing the immune response of an individual to delivery of gRNA, comprising administering any one of the gRNA disclosed herein, wherein the gRNA produces a reduced response by the immune system of the individual after administration. In some embodiments, the present invention includes a method for reducing activation in the immune system of an individual after administration compared to a control gRNA that has not been similarly modified.

[0699] In some embodiments, administration of Cas RNP or Cas9 mRNA together with a modified gRNA (e.g., sgRNA or dgRNA) produces lower serum concentrations of immunocytokines compared to administration of unmodified sgRNA. In some embodiments, the invention comprises a method of reducing serum concentrations of immunocytokines in an individual, comprising administering any of the gRNAs disclosed herein, wherein the gRNA produces a lower concentration of an immunocytokine in the serum of the individual compared to a control gRNA that is not similarly modified.

[0700] This specification and the exemplary embodiments should not be considered as limiting. For the purpose of this specification and the appended claims, unless otherwise stated, all numbers used in the specification and claims to represent quantities, percentages or ratios and other numerical values ​​should be understood to be modified by the term "about" in all cases, to the extent that it has not been so modified. Therefore, unless otherwise indicated, the numerical parameters listed in this specification and the appended claims are approximate values, which can vary depending on the desired properties attempted to be obtained. At least, it is not intended to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying general rounding techniques.

[0701] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," as well as any singular use of any word, include plural referents unless expressly and unambiguously limited to one referent. As used herein, the term "include" and its grammatical variations are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. Example

[0702] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the invention in any way.

[0703] Example 1 - Materials and Methods

[0704] A. Synthetic guide RNA (gRNA)

[0705] gRNAs were chemically synthesized by commercial suppliers in both dual guide (dgRNA, i.e., crRNA and trRNA) and single guide (sgRNA) formats with modified nucleotides and linkage patterns as provided in Table 4.

[0706] B. In vitro transcription of Cas9 mRNA (“IVT”)

[0707] Capped and polyadenylated Cas9 mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing T7 promoter and 100 nucleotides (nt) poly (A / T) region was linearized by XbaI and obtained from a commercial manufacturer. IVT reactions for generating Cas9 modified mRNA were incubated at 37°C for 4 hours under the following conditions: 50 ng / μL linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U / μL T7 RNA polymerase (NEB); 1 U / μL mouse RNase inhibitor (NEB); 0.004 U / μL inorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer. After the 4-hour incubation, TURBO deoxyribonuclease (ThermoFisher) was added to a final concentration of 0.01 U / μL, and the reaction was incubated for an additional 30 minutes to remove the DNA template. Cas9 mRNA was purified from enzyme and nucleotides using standard protocols, including silica-bound columns such as the MegaClear Transcription Clean-up Kit (ThermoFisher) or a precipitation step using LiCl followed by EtOH and NaOAc. Transcript concentrations were determined by measuring light absorption at 260 nm (Nanodrop), and transcripts were analyzed by capillary electrophoresis with a Bioanlayzer (Agilent).

[0708] C. Cas9 mRNA and gRNA transfection in Neuro2A cells

[0709] Mouse cell line Neuro2A was cultured in DMEM medium supplemented with 10% calf serum and plated in 96-well plates at a density of 15,000 cells per well 24 hours before transfection. On the day of transfection, the medium was aspirated from the cells and replaced with fresh medium. Lipofectamine-2000 (Invitrogen) was diluted 1:50 (v / v) in Opti-MEM (Invitrogen). Cas9 mRNA and single guide RNA were diluted separately in Opti-MEM. For the dual-guide format, crRNA and trRNA were diluted together in Opti-MEM at a 1:1 molar ratio. Cas9 mRNA and gRNA were mixed separately with diluted lipofectamine-2000 at 1:1 (v / v) to produce two lipid complexes. After incubation for 5 minutes, the lipid complexes were added to the cells continuously to obtain a final concentration of 100ngCas9 mRNA / well and 0.4μL total lipid particle transfection reagent. Two dose levels were tested for each experiment, including 25 nM and 2.5 nM, 16.7 nM and 1.67 nM, 10 nM and 1 nM, 8.3 nM and 0.83 nM, and 3 nM and 0.3 nM. For the dual guide, this concentration included equimolar amounts of crRNA and trRNA, such that, for example, 25 nM crRNA and 25 nM trRNA produced 25 nM total dual guide. Cells were lysed 24 hours after transfection, and the lysates were used directly for PCR reactions for editing analysis by NGS.

[0710] Cas9 mRNA with 1xNLS (SEQ ID NO: 359):

[0711]

[0712]

[0713]

[0714] Cas9 miRNA with 2xNLS and HA tags (SEQ ID NO: 360):

[0715]

[0716]

[0717] D. Primary hepatocytes

[0718] Primary mouse hepatocytes (PMH) (Gibco) were cultured according to the manufacturer's protocol (Invitrogen, protocol 11.28.2012). Briefly, cells were thawed and resuspended in hepatocyte thawing medium (Gibco, catalog number CM7000) with supplements, followed by centrifugation at 100 g for 10 minutes. The supernatant was discarded and the pelleted cells were resuspended in hepatocyte plating medium plus supplement packs (Invitrogen, catalog number A1217601 and CM3000). Cells were counted and plated on Bio-coat collagen I coated 96-well plates (ThermoFisher, catalog number 877272) at a density of 15,000 cells per well and incubated at 37° C. and 5% CO2 for 5 hours to allow monolayer formation. After 5 hours, the plating medium was removed and replaced with supplemented hepatocyte medium (Invitrogen, catalog number A1217601 and CM4000) containing LNP-formulated Cas9 mRNA and guide RNA plus 3% mouse serum. LNPs were diluted from a starting dose level of 100 ng Cas9 mRNA and approximately 30 nM guide RNA per well, and serial dilutions were performed until 0.1 ng mRNA and 0.03 nM guide RNA per well. Cells were incubated at 37° C. and 5% CO2 atmosphere for approximately 48 hours, followed by cell lysis and NGS analysis as described herein.

[0719] E. Lipid Nanoparticle ("LNP") Formulations

[0720] The LNPs were formulated with a cationic lipid amine to RNA phosphate (N:P) molar ratio of approximately 4.5. The lipid nanoparticle components are dissolved in 100% ethanol in the following molar ratios: 45 mol% (12.7 mM) cationic lipid (e.g., (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecadienoate, also known as 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)hydroxy)oxy)methyl)propyl (9Z,12Z)-octadecadienoate); 44 mol% (12.4 mM) auxiliary lipid (e.g., cholesterol); 9 mol% (2.53 mM) neutral lipid (e.g., DSPC); and 2 mol% (.563 mM) PEG (e.g., PEG2k-DMG). RNA cargo was prepared in 25 mM sodium acetate buffer, pH 4.5, resulting in an RNA cargo concentration of approximately 0.45 mg / mL.

[0721] LNPs were prepared by using a Precision Nanosystems NanoAssemblr according to the manufacturer's protocol. TM The benchtop instrument mixes lipid and RNA solutions microfluidically to form a 2:1 ratio of aqueous phase to organic solvent during mixing using differential flow rates.

[0722] LNP preparation scheme A: After mixing, LNPs were collected, diluted in phosphate buffered saline (PBS, approximately 1:1), and then the remaining buffer was exchanged into PBS (100-fold excess of sample volume) at 4°C with gentle stirring overnight using a 10 kDa Slide-a-Lyzer™ G2 dialysis cassette (ThermoFisher Scientific). LNPs were concentrated using a 10 kDa Amicon spin filter (centrifuged at 4000 g at 4°C) to achieve the desired concentration. The resulting mixture was then filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at 2-8°C.

[0723] LNP formulation protocol B: After mixing, LNPs were collected, diluted in 50 mM Tris, pH 7.5 (approximately 1:1), and then exchanged into 50 mM Tris, pH 7.5 (100-fold excess of sample volume) at 4°C with gentle agitation overnight using 10 kDa Slide-a-Lyzer™ G2 dialysis cassettes (ThermoFisher Scientific). LNPs were concentrated using 10 kDa Amicon spin filters (centrifuged at 4000 g at 4°C) to achieve twice the desired concentration. These concentrated LNPs were mixed 1:1 with 50 mM Tris, 90 mM NaCl, 10% sucrose (2×TSS), pH 7.5. The resulting mixture was then filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at -80°C.

[0724] LNP Formulation Scheme C: RNA cargo was prepared in 25 mM sodium citrate, 100 mM sodium chloride at pH 5, resulting in a concentration of approximately 0.45 mg / mL of RNA cargo. After mixing, LNPs were collected in water at a ratio of 3:1. LNPs were incubated at room temperature for one hour and mixed with water 1:1. They were then buffer exchanged into 1×TSS (50 mM Tris, 45 mM NaCl, 5% sucrose at pH 7.5) on a PD-10 column (GE Healthcare) using the manufacturer's protocol. LNPs were concentrated using a 10 kDa Amicon spin filter (centrifuged at 4000 g at 4°C) to achieve the desired concentration. The resulting mixture was then filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at -80°C.

[0725] F. Next-generation sequencing ("NGS") and analysis for hit cleavage efficiency

[0726] To quantitatively determine the editing efficiency at target locations in the genome, deep sequencing is used to identify the presence of insertions and deletions introduced by gene editing.

[0727] Design PCR primers around the target site (e.g., TTR, FVII) and amplify the genomic region domain of interest. Primer sequences are provided in Table 5 below.

[0728] Table 5

[0729]

[0730] Additional PCR was performed according to the manufacturer's protocol (Illumina) to add the required chemistry for sequencing. Amplicons were sequenced on an Illumina MiSeq instrument. After eliminating reads with low quality scores, the reads were aligned to a human reference genome (e.g., hg38). The resulting archive containing the reads was mapped to a reference genome (BAM archive), where reads overlapping the target region domain of interest were selected and the number of wild-type reads relative to the number of reads containing insertions, substitutions, or deletions were calculated.

[0731] The editing percentage (eg, "editing efficiency" or "editing %") is defined as the total number of sequence reads with an insertion or deletion compared to the total number of sequence reads that include the wild type.

[0732] G. In vivo LNP delivery

[0733] CD-1 female mice in the range of 6-10 weeks of age were used for each study. Animals were weighed and grouped according to body weight to prepare the administration solution based on the group average body weight. LNP was administered via the lateral tail vein in a volume of 0.2 mL (approximately 10 mL per kg of body weight) per animal. Approximately 6 hours after administration, the animals were observed for side effects. Body weight was measured at 24 hours after administration, and the animals were euthanized at various time points by bleeding via cardiac puncture under isoflurane anesthesia. Blood was collected in serum separation tubes or in tubes containing buffered sodium citrate for plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the mid lobe from each animal for DNA extraction and analysis.

[0734] H. Cytokine Induction Analysis

[0735] For this analysis, approximately 50-100 μ L blood is collected by tail vein incision for serum cytokine measurement. Make blood coagulate approximately 2 hours at room temperature, and then centrifuge 10 minutes at 1000xg, then collect serum. Measure IL-6, TNF-α, IFN-α and MCP-1 based on Luminex magnetic bead multiplex analysis (Affymetrix ProcartaPlus, catalog number Exp040-00000-801) for collecting cytokine analysis in sample. Prepare kit reagents and standards as instructed in the manufacturer's scheme. 25 μ L mouse serum is added to the hole containing 25 μ L dilution antibody coated magnetic beads. Flat plate is incubated at room temperature for 2 hours and then washed. Diluted biotin antibody (50 μ L) is added to beads and incubated at room temperature for 1 hour. Wash beads again, then add 50 μ L dilution streptavidin-PE to each hole, then incubate for 30 minutes. The beads were washed again and then suspended in 100 μL of wash buffer and read on a Bio-Plex 200 instrument (Bio-Rad).The data were analyzed using the BioplexManager version 6.1 analysis package by using a five parameter logistic curve fit to cytokine concentrations calculated from the standard curve.

[0736] I. Genomic DNA Isolation

[0737] For in vivo studies, genomic DNA was extracted from 10 mg of tissue using a bead-based extraction kit MagMAX-96 DNA Multi-Sample Kit (ThermoFisher, catalog number 4413020) according to the manufacturer's protocol, which included homogenizing the tissue in lysis buffer (roughly 400 μL / 10 mg tissue). All DNA samples were standardized to a concentration of 100 ng / μL for PCR and subsequent NGS analysis, as described herein.

[0738] J. Transthyretin (TTR) ELISA analysis

[0739] Blood was collected and serum was separated as indicated. Total TTR serum levels were determined using a mouse prealbumin (transthyretin) ELISA test kit (Aviva Systems Biology, catalog number OKIA00111). Kit tests and standards were prepared according to the manufacturer's protocol. Mouse serum was diluted to a final dilution of 10,000 times by 1× assay diluent. This was performed as follows: two consecutive 50-fold dilutions were performed, resulting in a 2500-fold dilution. A final 4-fold dilution step was performed so that the total sample dilution was 10,000 times. Standard curve dilutions (100 μL each) and diluted serum samples were added to each well of an ELISA plate pre-coated with capture antibody. The plate was incubated at room temperature for 30 minutes and then washed. Enzyme-antibody conjugate (100 μL / well) was added for 20 minutes of incubation. Unbound antibody conjugates were removed and the plate was washed again before adding the chromogenic substrate solution. The plate is incubated for 10 minutes, followed by the addition of 100 μL of stop solution, such as sulfuric acid (approximately 0.3 M). The plate is read at an absorbance of 450 nm on a SpectraMax M5 plate reader. Serum TTR levels are calculated by SoftMaxPro software version 6.4.2 using a four-parameter logistic curve fit off the standard curve. Final serum values ​​are adjusted for analytical dilution.

[0740] Example 2 - Engineering of modified gRNA and in vitro testing

[0741] Modified gRNAs were designed in a dual-guide format (dgRNA) as shown in Table 4. Thus, both modified crRNAs and trRNAs were designed and chemically synthesized to allow for the pairing of modified and unmodified components that form dgRNAs. These pairs were transfected into Neuro2A cells at concentrations as indicated in the figure and the editing efficiency (e.g., % editing) was measured by NGS as described in Example 1.

[0742] Certain modified crRNAs from Table 4 targeting the mouse TTR gene were transfected with Cas9 mRNA and unmodified trRNA (TR000002). The guide tested included SEQ ID Nos: 1 to 18. Figure 1 As shown in , some of the modified crRNAs (along with the unmodified trRNA) conferred similar or enhanced activity compared to the unmodified control, while other modified crRNAs reduced activity.

[0743] At the same time, the modified trRNA from Table 4 was transfected with Cas9 mRNA targeting the same sequence of the mouse TTR gene together with unmodified crRNA (CR000686). The guide tested included SEQ ID Nos: 188 to 200 and 204. Figure 2As shown in , many modified trRNAs (along with unmodified crRNAs) conferred similar or enhanced activities compared to unmodified controls, while some of the modified trRNAs reduced activity.

[0744] In addition to replacing chemically modified nucleotides, some of the crRNA and trRNA pairs tested were also engineered by sequence substitutions, such as generating GC pairs not found in the parental sequences. The tested sequences include SEQ ID Nos: 15 and 201; SEQ ID Nos: 16 and 202; SEQ ID Nos: 1 and 188. Figure 3 As shown in , one such pair (SEQ ID Nos: 16 and 202) produced similar or enhanced activity compared to the unmodified control, while both of the pair had reduced activity.

[0745] Subsequently, the pairing of the modified crRNA and modified trRNA from Table 4 was tested. Figure 4 As shown in , some of the pairs of modified crRNA and modified trRNA conferred similar or enhanced activity compared to the unmodified control, while some of the pairs reduced activity. Figure 4 In the figure, the header row depicts the different trRNAs used in the experiment, and the header column depicts the different crRNAs used. To determine the combination used in the experiment, the rows and columns are matched. TR000002 and CR000686 are unmodified controls (see the lower right cell).

[0746] Based on the dgRNA design, the corresponding single guide RNA (sgRNA) was modified to have some of the features (aspects) of the modified crRNA and trRNA, as shown in Table 4 and Fig.15D These sgRNAs, SEQ ID Nos: 228 to 234, were also tested in Neuro2A cells and as shown in Figure 5 As shown in , each of the modified sgRNAs showed similar activity to the control (G0000209; SEQ ID NO: 228) containing only 5' and 3' end modifications.

[0747] For Table 4 and Figure 6 A similar set of experiments was performed with additional dgRNA guides as described in . Guides tested included SEQ ID Nos: 32 to 47 and 1. Modified crRNAs also targeting the mouse TTR gene were transfected with Cas9 mRNA and unmodified trRNA (TR000002). Figure 6 As shown in , some modified crRNAs (along with unmodified trRNAs) conferred similar or enhanced activity compared to an unmodified control (CR000686), while other modified crRNAs reduced activity.

[0748] At the same time, if Figure 7 As shown in , the modified trRNA from Table 4 was transfected with Cas9 mRNA targeting the same sequence of the mouse TTR gene together with unmodified crRNA (CR000686). The guide tested included SEQ ID Nos: 205 to 222 and 1. Figure 7 As shown in , many modified trRNAs (along with unmodified crRNA) conferred similar or enhanced activities compared to the unmodified control (TR000002), while some of the modified trRNAs reduced activity.

[0749] In addition to replacing chemically modified nucleotides, some of the tested crRNA and trRNA pairs from Table 4 were also engineered by sequence substitutions, such as to generate GC pairs or GU mismatches ("GU wobble") not found in the parental sequence. Figure 8 As shown in , some of the modifications and pairings conferred similar or enhanced activity compared to the unmodified control, while some (eg, "GU wobble" or mismatched pairings) reduced activity. Figure 8 The results using the trRNA guides shown in SEQ ID Nos: 223 to 227 and 188 and the crRNA guides shown in SEQ ID Nos: 48 to 52 and 1 are shown.

[0750] Then, if Fig. 9 Selected pairs of modified crRNA and modified trRNA from Table 4 were tested as shown in . Some of the pairs of modified crRNA and modified trRNA conferred similar or enhanced activity compared to the unmodified control, while some of the pairs reduced activity. Fig. 9 In the , the header row depicts the different trRNAs used in the experiment, and the header column depicts the different crRNAs used. To determine the combination used in the experiment, the rows and columns were matched. The unmodified controls were TR000002 and CR000686.

[0751] Some of the modified gRNAs (dgRNA and sgRNA) from Table 4 were also tested in purely biochemical assays (i.e., cell-free cleavage assays). Interestingly, many of the modified gRNAs that were largely inactive in Neuro2A cells were active in the biochemical assays, indicating that such biochemical assays may not predict cell-mediated activity of modified gRNAs (data not shown).

[0752] Example 3. Further testing of modified gRNAs for other targets

[0753] Having determined that certain modifications affect gRNA activity, it was subsequently tested whether these modifications would affect activity when targeting (1) independent sequences in the same gene or (2) sequences in different genes. Therefore, gRNAs targeting another sequence in the mouse TTR gene and a sequence in the mouse Factor-VII (FVII) gene were engineered and synthesized to have certain modification patterns tested in Example 2 (see Table 4). These gRNAs were transfected into Neuro2A cells at concentrations as indicated in the figure and the editing efficiency (e.g., % editing) was measured by NGS as described in Example 1.

[0754] Modified crRNAs from Table 4 targeting the mouse TTR gene (different sequences than those targeted in Example 2) or the mouse FVII gene were transfected with Cas9 mRNA and unmodified trRNA (TR000002). Instructions for testing include Fig. 12A and 12B Some modified crRNAs (along with unmodified trRNAs) confer similar or enhanced activity compared to unmodified controls, while other modified crRNAs reduce activity.

[0755] At the same time, the modified trRNA from Table 4 was transfected with Cas9 mRNA targeting the same sequence of the mouse TTR gene (CR000705; a different sequence than that targeted in Example 2) or the same sequence as the mouse FVII gene (CR000657) together with unmodified crRNA. Fig.13A and 13B As shown in, compared to the unmodified control, many modified trRNAs (together with unmodified crRNAs) confer similar or enhanced activity, while some of the modified trRNAs reduce activity. This data shows that certain modification patterns tend to have similar effects through different sequences.

[0756] Based on the dgRNA design described above, the corresponding single guide RNA (sgRNA) was modified to have the aspect of some of the modified crRNA and trRNA. See Table 4. These sgRNAs were also tested in Neuro2A cells. The results are shown in Fig.10 (mouse TTR) and Fig.11 (mouse FVII). These experiments show that some modification patterns produce similar effects even when targeting different genes.

[0757] Example 4. In vivo testing of modified gRNA

[0758] Following in vitro testing, the modified sgRNAs were delivered to animals in six independent studies to determine whether the modifications conferred any benefits for editing in vivo.

[0759] LNPs were formulated with IVTCas9 mRNA together with chemically modified sgRNA (targeting TTR or FVII) as described in Example 1. The ratio of mRNA:sgRNA was approximately 1: 1 by weight of the RNA components. Unless otherwise specified, the Cas9 mRNA used in the studies described in this example had the sequence of SEQ ID NO: 360 and LNPs were formulated using LNP formulation protocol A described above.

[0760] In one experiment, a single dose of LNP was administered to mice (n=5 per group) at 2 mg / kg and blood was collected four hours after dosing for serum cytokine analysis. Seven days after dosing, liver and blood were collected at necropsy for NGS measurement of editing efficiency and serum TTR analysis, respectively. Each of the sgRNAs in this experiment targeted the same sequence in the TTR gene, and the only difference between the sgRNAs was the modification made to each (see Fig.14A to D and 15A to E; Table 4 SEQ ID No: 228 to 234). G000209 (two batches tested) served as a less modified control, with only 2'-O-methyl modifications and phosphorothioate bonds at and between the three terminal nucleotides at the 5' and 3' ends of the sgRNA, respectively. (See Fig.15D ).

[0761] Fig.14A The results presented in Figures 4 to 5 show that the more modified sgRNAs tended to induce lesser responses for each of the analyzed cytokines compared to the less modified G000209 control. The more modified sgRNAs also conferred greater editing efficiency in the livers of treated animals, with the % editing of two of the more modified sgRNAs (e.g., G000263 and G000267) reaching approximately 60% ( 44-47% for the less modified control (G000209 batch) compared to approximately 44-47% for the less modified control (G000209 batch). Fig.15A Importantly, editing efficiency correlated with phenotypic changes, as serum knockdown of TTR levels was comparable to or significantly greater than less modified controls (see e.g. Figures 15A to 15B The differences between the terminally modified G000209 and the highly modified G000267 are summarized in Fig.15D and 15E (2'-O-Me modified nucleotides are shown in bold, and * indicates phosphorothioate bonds).

[0762] In another in vivo study, three sgRNAs targeting independent sequences in the mouse TTR gene were tested. A single dose of LNP was administered to mice (n=5 per group) at 2 mg / kg, 1 mg / kg, or 0.3 mg / kg. Blood was collected four hours after dosing for serum cytokine analysis. Seven days after dosing, liver and blood were collected at necropsy for NGS measurement of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene (a different sequence from that targeted in the previous in vivo study), with one sgRNA being completely unmodified (G000201 (SEQ ID NO: 243)), another having only terminal modifications (G000211 (SEQ ID NO: 241)), having 2'-O-methyl modifications and phosphorothioate bonds at and between the three terminal nucleotides at the 5' and 3' ends of the sgRNA, respectively), and the third sgRNA having the same modification pattern as G000267 in the previous in vivo study (G000282 (SEQ ID NO: 242)).

[0763] like Figures 16A to 16D As shown in , each of the sgRNAs produced similar responses to each of the tested cytokines in a dose-dependent manner. For editing efficiency, the unmodified sgRNA (G000201 (SEQ ID NO: 243)) conferred minimal in vivo editing, while the largely modified sgRNA (G000282 (SEQ ID NO: 242)) conferred a level of approximately 60% at a dose of 2 mg / kg, which was significantly greater than the level achieved with the less modified sgRNA (G000211 (SEQ ID NO: 241)) ( Fig.17A and B). As in the previous in vivo studies, editing levels correlated with the amount of serum TTR knockdown ( Fig. 17C and D).

[0764] A study similar to the second in vivo study was then performed with a set of three additional sgRNAs targeting different TTR sequences in the mouse TTR gene (targeting sequences different from those targeted in the two previous in vivo studies). A single dose of LNP was administered to mice (n=5 per group) at 2 mg / kg, 1 mg / kg, or 0.3 mg / kg. Blood was collected four hours after dosing for serum cytokine analysis. Seven days after dosing, liver and blood were collected at necropsy for NGS measurement of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene (a different sequence from that targeted in the two previous in vivo studies), with one sgRNA being completely unmodified (G000285; (SEQ ID NO: 332)), another having only terminal modifications (G000269 (SEQ ID NO: 330)), having 2'-O-methyl modifications and phosphorothioate bonds at and between the three terminal nucleotides at the 5' and 3' ends of the sgRNA, respectively), and the third sgRNA having the same modification pattern as G000267 and G000282 in the two previous in vivo studies (G000283 (SEQ ID NO: 331)).

[0765] In this study, the unmodified sgRNA (G000285 (SEQ ID NO: 332)) conferred minimal in vivo editing, while the largely modified sgRNA (G000283 (SEQ ID NO: 331)) conferred levels reaching approximately 60% at a dose of 2 mg / kg, which was significantly greater than the levels achieved with the less modified sgRNA (G000269 (SEQ ID NO: 330)). Figures 18A to 18B As in the previous in vivo studies, editing levels correlated with the amount of serum TTR knockout ( Fig.18C ).

[0766] In the fourth in vivo study, the effect of modification of gRNA was evaluated for another gene (FVII). For comparison within the study, two of the sgRNAs tested in the first in vivo study (G000209 and G000267) were included. A single dose of LNP was administered to mice (n=5 per group) at 2 mg / kg, 1 mg / kg or 0.3 mg / kg and blood was collected four hours after administration for serum cytokine analysis. Six days after administration, livers were collected at autopsy for NGS measurements of editing efficiency. In this study, each of the sgRNAs targeted the same sequence in the TTR or FVII gene, with one sgRNA for each gene having only terminal modifications, G000208 (SEQ ID NO: 286) for FVII and G000209 for TTR, both of which have 2'-O-methyl modifications and phosphorothioate bonds at and between the three terminal nucleotides at the 5' and 3' ends of the sgRNA, respectively), and the second sgRNA having the same modification pattern as G000267, G000282, and G000283 in the previous in vivo study for FVII (G000373 (SEQ ID NO: 287); G000267 (SEQ ID NO: 234) for TTR).

[0767] like Figures 19A to 19D As shown in , each of the sgRNAs produced similar responses in a dose-dependent manner for each of the tested cytokines. For editing efficiency, the editing efficiency of the more modified sgRNA targeting FVII (G000373 (SEQ ID NO: 287)) increased at each of the doses tested compared to the less modified version (G000208 (SEQ ID NO: 286)). Fig.18A ). These results were also observed for sgRNA targeting TTR ( Figures 20A to 20B ).

[0768] In another in vivo study, ten additional sgRNAs that target the same sequence in the mouse TTR gene as G000282 were tested. G000282 was also included in the study for comparison purposes. A single dose of LNP was administered to mice (n=5 per group) at 1 mg / kg or 0.5 mg / kg. The LNPs used in this study were formulated using LNP formulation scheme B described above. Seven (7) days after administration, liver and blood were collected at autopsy for NGS measurement of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targets the same sequence in the TTR gene. The modification patterns of each sgRNA tested varied and included 2'-OMe, 2'-F, and PS modifications in the 5' terminal region, 3' terminal region, hairpin 1 region, hairpin 2 region, connecting region, lower stem region, protrusion region, and upper stem region of the sgRNA. The results of this study are shown in Figures 22A to 22C Including editing %( Fig.22A ), mean edit and standard deviation ( Fig. 22B ) and serum TTR levels ( Fig. 22C ). These same sgRNAs were tested in primary mouse hepatocytes according to the methods described herein. The results of this dose-response TTR editing study are shown in Figures 24A to 24C Including editing %( Fig.24A ), dose response curve ( Fig. 24B ) and EC50 value ( Fig.24C ).

[0769] In another in vivo study, thirteen additional sgRNAs that target the same sequence in the mouse TTR gene as G000282 were tested. G000282 was also included in the study for comparison purposes. A single dose of LNP was administered to mice (n=5 per group) at 1 mg / kg. The LNPs used in this study were formulated using LNP formulation scheme C described above. The Cas9 mRNA used in this study has the sequence of SEQ ID NO: 359. Blood was collected four hours after dosing for serum cytokine analysis. Seven days after dosing, liver and blood were collected at autopsy for NGS measurements of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene. The sgRNAs tested included additional 2'-OMe and PS modifications in the 5' terminal region, 3' terminal region, hairpin 1 region, hairpin 2 region, and upper stem region of the sgRNA. The results of this study are shown in Figures 23A-23C Including editing %( Fig.23A ), average editing %( Fig. 23B ) and serum TTR levels ( Fig.23C ).

[0770] Some embodiments of the present disclosure:

[0771] 1. A single guide RNA comprising a 5' end modification and one or more of the following modifications:

[0772] a. Upper stem area;

[0773] b. Hairpin 1 area; and

[0774] c. Hairpin 2 area,

[0775] wherein the 5' end modification comprises at least two phosphorothioate (PS) bonds within the first seven nucleotides at the 5' end of the 5' terminus.

[0776] 2. The sgRNA of embodiment 1, wherein at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0777] 3. The sgRNA of embodiment 1 or embodiment 2, wherein at least one modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[0778] 4. The sgRNA of any one of embodiments 1 to 3, wherein at least one modification comprises a phosphorothioate (PS) bond between nucleotides.

[0779] 5. The sgRNA of any one of embodiments 1 to 4, wherein the sgRNA comprises one or more modifications in the upper stem region.

[0780] 6. The sgRNA of embodiment 5, comprising modifications at US1 to US12.

[0781] 7. The sgRNA of any one of embodiments 1 to 6, wherein the sgRNA comprises one or more modifications in the hairpin 1 region.

[0782] 8. The sgRNA of embodiment 7, wherein the sgRNA comprises a modification at H1-1.

[0783] 9. The sgRNA of any one of embodiments 1 to 8, wherein the sgRNA comprises one or more modifications in the hairpin 2 region.

[0784] 10. The sgRNA of embodiment 9, wherein the sgRNA comprises a modification at H2-1.

[0785] 11. The sgRNA of any one of embodiments 1 to 10, wherein the sgRNA comprises modifications at H1-1 to H1-12.

[0786] 12. The sgRNA of any one of embodiments 1 to 11, wherein the sgRNA comprises modifications at H2-1 to H2-15.

[0787] 13. The sgRNA of any one of embodiments 1 to 12, wherein the sgRNA comprises one or more modifications in each of the upper stem region, the hairpin 1 region, and the hairpin 2 region.

[0788] 14. The sgRNA of any one of embodiments 1 to 13, wherein the sgRNA comprises modified nucleotides between the hairpin 1 region and the hairpin 2 region.

[0789] 15. The sgRNA of any one of embodiments 1 to 14, further comprising a lower stem region comprising a modification.

[0790] 16. The sgRNA of any one of embodiments 1 to 15, further comprising a 3' terminal region comprising a modification.

[0791] 17. The sgRNA of embodiment 16, further comprising a 3' end modification in the 3' end.

[0792] 18. The sgRNA of embodiment 17, wherein at least two of the last four nucleotides at the 3' end of the 3' terminus are modified.

[0793] 19. The sgRNA of embodiment 17, wherein at least two of the last four nucleotides at the 3' end of the 3' terminus are modified with 2'-O-Me, 2'-F or 2'-O-moe.

[0794] 20. The sgRNA of any one of embodiments 17 to 19, further comprising a phosphorothioate (PS) bond between one or more of the last four nucleotides at the 3' end of the 3' terminus.

[0795] 21. The sgRNA of any one of embodiments 1 to 20, further comprising a protrusion region comprising a modification.

[0796] 22. The sgRNA of any one of embodiments 1 to 21, further comprising a linker region comprising a modification.

[0797] 23. The sgRNA of any one of embodiments 1 to 22, wherein at least the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus are modified.

[0798] 24. The sgRNA of any one of embodiments 1 to 23, wherein the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus are linked to phosphorothioate (PS) bonds.

[0799] 25. The sgRNA of embodiment 24, wherein the terminal modification comprises 2'-O-Me.

[0800] 26. The sgRNA of embodiment 24, wherein the terminal modification comprises 2'-F.

[0801] 27. The sgRNA of any one of embodiments 1 to 26, wherein the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus are linked to a PS bond, and wherein the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus comprise a 2'-O-Me modification.

[0802] 28. The sgRNA of any one of embodiments 1 to 26, wherein the first four nucleotides at the 5' terminus and the last four nucleotides at the 3' terminus are linked to a PS bond, and wherein the first three nucleotides at the 5' terminus and the last three nucleotides at the 3' terminus comprise 2'-O-Me, 2'-F and / or 2'-O-moe modifications.

[0803] 29. The sgRNA of any one of embodiments 1 to 28, wherein LS1, LS6, LS7, LS8, LS11 and / or LS12 are modified with 2'-O-Me.

[0804] 30. The sgRNA of any one of embodiments 1 to 29, wherein each of the nucleotides in the protuberance region is modified with 2'-O-Me.

[0805] 31. The sgRNA of any one of embodiments 1 to 29, wherein at least 50% of the nucleotides in the protuberance region are modified with 2'-O-Me.

[0806] 32. The sgRNA of any one of embodiments 1 to 31, wherein each of the upper stem region nucleotides is modified with 2'-O-Me.

[0807] 33. The sgRNA of any one of embodiments 1 to 32, wherein N16, N17 and / or N18 in the linker region are modified with 2′-O-Me.

[0808] 34. The sgRNA of any one of embodiments 1 to 32, wherein N15, N16, N17 and / or N18 in the joining region are modified.

[0809] 35. The sgRNA of embodiment 33 or embodiment 34, wherein the modification in the linker region is selected from 2'-O-Me and 2'F.

[0810] 36. The sgRNA of any one of embodiments 32 to 35, wherein N16, N17 and N18 are linked to PS bonds.

[0811] 37. The sgRNA of any one of embodiments 1 to 36, wherein each of the nucleotides in the hairpin 1 region is modified with 2'-O-Me.

[0812] 38. The sgRNA of any one of embodiments 1 to 37, wherein each of the nucleotides in the hairpin 2 region is modified with 2'-O-Me.

[0813] 39. A single guide RNA (sgRNA) comprising 2'-O-Me modified nucleotides at the following positions:

[0814] a. The first three nucleotides at the 5' end of the 5' terminus;

[0815] b. LS1, LS6, LS7, LS8, LS11 and / or LS12 in the lower stem region;

[0816] c. B1 and / or B2 in the protrusion area;

[0817] d. Each nucleotide in the upper stem region;

[0818] e. N16, N17 and / or N18 in the connecting region;

[0819] f. Each nucleotide in the hairpin 1 region;

[0820] g. each nucleotide in region 2 of the hairpin; and

[0821] h. Last four nucleotides at the 3' end.

[0822] 40. The sgRNA of embodiment 39, wherein B3 to B6 are modified with 2'-O-Me.

[0823] 41. The sgRNA of embodiment 39. It further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0824] 42. The sgRNA of any one of the preceding embodiments, wherein LS9 and LS10 are modified, for example, with 2'-F or 2'-OMe.

[0825] 43. The sgRNA of any one of the preceding embodiments, wherein N15, N16, N17 and N18 are modified, for example, with 2'-F or 2'-OMe.

[0826] 44. The sgRNA of any of the preceding embodiments, wherein H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 are modified with 2'-F.

[0827] 45. The sgRNA of any one of the preceding embodiments, wherein the second to last, third to last, and fourth to last nucleotides at the 3' end are modified with 2'-F.

[0828] 46. ​​A single guide RNA (sgRNA) comprising a 2'-F modified nucleotide at the following position:

[0829] a. LS9 and LS10 in the lower stem region;

[0830] b. N15, N16, N17 and N18 in the connection region; and

[0831] c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14 and H2-15 in the hairpin 2 region.

[0832] 47. The sgRNA of embodiment 46, further comprising 2'-F modified nucleotides at the second to last, third to last, and fourth to last nucleotides at the 3' end.

[0833] 48. The sgRNA of embodiment 46 or embodiment 47, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0834] 49. The sgRNA of any one of embodiments 46 to 48, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end of the 5' end and 2'-O-Me or 2'-F modified nucleotides at three of the last four nucleotides at the 3' end of the 3' end.

[0835] 50. A single guide RNA (sgRNA) comprising:

[0836] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0837] b. 2'-O-Me modified nucleotides at US1 to US12;

[0838] c. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0839] d. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0840] e. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0841] 51. The sgRNA of embodiment 50, further comprising 2'-O-Me modified nucleotides at LS1 and / or LS6.

[0842] 52. The sgRNA of embodiment 50 or embodiment 51, further comprising 2'-O-Me modified nucleotides between the hairpin 1 region and the hairpin 2 region.

[0843] 53. A single guide RNA (sgRNA) comprising:

[0844] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0845] b. 2'-F modified nucleotides at LS1 to LS6;

[0846] c. 2'-O-Me modified nucleotides at US1 to US12;

[0847] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0848] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0849] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0850] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0851] 54. A single guide RNA (sgRNA) comprising:

[0852] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0853] b. 2'-F modified nucleotides at LS2 to LS5;

[0854] c. 2'-O-Me modified nucleotides at LS1 and LS6;

[0855] d. 2'-O-Me modified nucleotides at US1 to US12;

[0856] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0857] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0858] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0859] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0860] 55. A single guide RNA (sgRNA) comprising

[0861] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0862] b. 2'-O-Me modified nucleotides at US1 to US12;

[0863] c. 2'-O-Me modified nucleotides at LS7, LS8, LS11 and LS12;

[0864] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0865] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0866] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0867] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0868] 56. A single guide RNA (sgRNA) comprising

[0869] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0870] b. 2'-O-Me modified nucleotides at US1 to US12;

[0871] c. 2'-O-Me modified nucleotides at LS7, LS8, LS11 and LS12;

[0872] d. 2'-F modified nucleotides at LS9 and LS10;

[0873] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0874] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0875] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0876] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0877] 57. A single guide RNA (sgRNA) comprising

[0878] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0879] b. 2'-O-Me modified nucleotides at US1 to US12;

[0880] c. 2'-O-Me modified nucleotides at LS8, LS10 and LS12;

[0881] d. 2'-OF modified nucleotides at LS7, LS9 and LS11;

[0882] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0883] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0884] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0885] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0886] 58. A single guide RNA (sgRNA) comprising:

[0887] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0888] b. 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11 and LS12

[0889] c. 2'-O-Me modified nucleotides at US1 to US12;

[0890] d. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0891] e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0892] f. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0893] g. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0894] 59. A single guide RNA (sgRNA) comprising

[0895] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0896] b. 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11 and LS12;

[0897] c. 2'-F modified nucleotides at LS9 and LS10;

[0898] d. 2'-O-Me modified nucleotides at US1 to US12;

[0899] e. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0900] f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0901] g. 2'-O-Me modified nucleotides at H2-1 to H2-15; and

[0902] h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end of the 3' terminus.

[0903] 60. A single guide RNA (sgRNA) comprising

[0904] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0905] b. 2'-O-Me modified nucleotides at US1 to US12;

[0906] c. 2'-O-Me modified nucleotides at H1-1 to H1-12;

[0907] d. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2;

[0908] e. 2'-O-Me modified nucleotides at H2-1 to H2-8;

[0909] f. 2'-F modified nucleotides at H2-9 to H2-15;

[0910] g. 2'-F modified nucleotides at the second to last, third to last and fourth to last nucleotides at the 3' terminus; and

[0911] h. 2'-O-Me modified nucleotide at the last nucleotide at the 3' end of the 3' terminus.

[0912] 61. A single guide RNA (sgRNA) comprising

[0913] a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end of the 5' terminus;

[0914] b. 2'-O-Me modified nucleotides at US1 to US12;

[0915] c. 2'-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12;

[0916] d. 2'-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9 and H1-11;

[0917] e. 2'-F modified nucleotides between hairpin 1 and hairpin 2;

[0918] f. 2'-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14;

[0919] g. 2'-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13 and H2-15;

[0920] h. 2'-F modified nucleotides at the second to last and fourth to last nucleotides at the 3' end; and

[0921] i. 2'-O-Me modified nucleotides at the third to last and last nucleotides at the 3' end of the 3' terminus.

[0922] 62. The sgRNA of any one of embodiments 50 to 61, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0923] 63. The sgRNA of any one of embodiments 50 to 61, further comprising at least one phosphorothioate (PS) bond within the first seven nucleotides at the 5' end of the 5' terminus.

[0924] 64. A single guide RNA (sgRNA) comprising

[0925] a. 2'-O-Me modified nucleotides at LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15; and

[0926] b. 2'-F modified nucleotides at LS7, LS9, LS11; H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12 and H2-14.

[0927] 65. The sgRNA of embodiment 64, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[0928] 66. The sgRNA of embodiment 64 or embodiment 65, further comprising

[0929] a. 2'-O-Me modified nucleotides at the last and third to last nucleotides at the 3' end of the 3' terminus; and

[0930] b. 2'-F modified nucleotides at the second to last and fourth to last nucleotides at the 3' end of the 3' terminus.

[0931] 67. An sgRNA comprising any one of SEQ ID Nos: 228 to 332, comprising the modifications of Table 4.

[0932] 68. An sgRNA comprising any one of SEQ ID Nos: 235 to 240, 265 to 285, and 309 to 329, comprising the modification of Table 4.

[0933] 69. sgRNA comprising SEQ ID No: 240.

[0934] 70. An sgRNA comprising SEQ ID No: 240, comprising the modifications of Table 4.

[0935] 71. An sgRNA comprising SEQ ID No: 242, comprising the modifications of Table 4.

[0936] 72. sgRNA comprising modifications of SEQ ID No: 242 as shown in Table 4.

[0937] 73. sgRNA comprising SEQ ID No: 358.

[0938] 74. An sgRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity to a nucleic acid of any one of SEQ ID Nos: 235 to 240, 265 to 285 and 309 to 329, wherein the modification at each nucleotide of the sgRNA corresponding to the nucleotide of the reference sequence identifier in Table 4 is the same or equivalent to the modification shown in the reference sequence identifier in Table 4.

[0939] 75. The sgRNA of any one of embodiments 66 to 74, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0940] 75. The sgRNA of any one of embodiments 1 to 74, further comprising at least three PS bonds connecting nucleotides in the hairpin 1 region.

[0941] 76. The sgRNA of any one of embodiments 1 to 75, further comprising at least three PS bonds connecting nucleotides in the hairpin 2 region.

[0942] 77. The sgRNA of any one of embodiments 1 to 76, further comprising at least three PS bonds connecting nucleotides in the upper stem region.

[0943] 78. The sgRNA of any one of embodiments 1 to 77, wherein the sgRNA forms a ribonucleoprotein complex with Streptococcus pyogenes Cas9.

[0944] 79. A guide RNA comprising a 2'-O-Me modification at each nucleotide in the upper stem region.

[0945] 80. A guide RNA comprising a 2'-O-Me modification at one, two, three or four, five, six, seven, eight, nine, ten or eleven of the following nucleotides: US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11 and US12.

[0946] 81. A guide RNA comprising 2'-O-Me modifications at 50% or more of the nucleotides in the hairpin 1 region and the hairpin 2 region.

[0947] 82. A guide RNA comprising a 2'-O-Me modification at each nucleotide in the upper stem region and a 2'-O-Me modification at 50% or more of the nucleotides in the hairpin 1 region and the hairpin 2 region.

[0948] 83. A guide RNA comprising or consisting of a 2'-O-Me modification starting at each nucleotide at H2-1 or H2-2 to the last nucleotide at the 3' terminus.

[0949] 84. A guide RNA comprising or consisting of a 2'-O-Me modification at LS1 and / or LS6 and no modification at LS2 to LS5.

[0950] 85. A guide RNA comprising 2'-O-Me modified nucleotides at 2 or more of LS8, LS9, LS10, LS11 and / or LS12.

[0951] 86. The guide RNA of embodiment 85, wherein at least LS8 and LS10 are modified with 2'-O-Me.

[0952] 87. The guide RNA of any one of embodiments 79 to 86, further comprising 2'-O-Me modifications at 50% or more of the nucleotides in the linker region.

[0953] 88. The guide RNA of embodiment 87, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the linker region are modified with 2'-O-Me.

[0954] 89. A guide RNA comprising 2'-O-Me modifications at up to 50% of the nucleotides in the linker region.

[0955] 90. A guide RNA comprising 2'-O-Me modifications at four or more nucleotides in a linker region.

[0956] 91. The guide RNA of embodiment 90, wherein at least one, two, three, four or five of nucleotides N2 to N6 in the linker region are modified with 2'-O-Me.

[0957] 92. A guide RNA comprising 2'-O-Me modified nucleotides at ten or more nucleotides in the linker region.

[0958] 93. The guide RNA of embodiment 92, wherein at least N2 to N6 are modified with 2'-O-Me.

[0959] 94. A guide RNA comprising 2'-O-Me modifications at 50% or more of the nucleotides in the protuberance region.

[0960] 95. The guide RNA of embodiment 94, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the protuberance region are modified with 2'-O-Me.

[0961] 96. The guide RNA of embodiment 94, wherein B2, B3 and / or B4 are modified with 2'-O-Me.

[0962] 97. A guide RNA comprising 2'-O-Me modified nucleotides at three or more nucleotides in the protuberance region.

[0963] 98. The guide RNA of embodiment 97, wherein B2, B3 and / or B4 are modified with 2'-O-Me.

[0964] 99. A guide RNA comprising the nucleotides of SEQ ID NO: 357, which has a modification pattern of the guide RNA as in any one of the preceding embodiments.

[0965] 100. A guide RNA comprising the nucleotide of SEQ ID NO: 356, which has a modification pattern of the guide RNA as in any one of the preceding embodiments.

[0966] 101. The guide RNA of any one of embodiments 79 to 100, further comprising a 2'-O-Me modification at the first three, four or five nucleotides at the 5' end of the 5' terminal region.

[0967] 102. The guide RNA of any one of embodiments 79 to 100, further comprising a 2'-O-Me modification at the last three, last four, or last five nucleotides at the 3' end of the 3' terminal region.

[0968] 103. The guide RNA of embodiment 102, wherein the last nucleotide at the 3' end of the 3' terminal region is unmodified.

[0969] 104. The guide RNA of embodiment 102, wherein only the second to last and third to last nucleotides at the 3' end of the 3' terminal region are modified.

[0970] 105. The guide RNA of embodiment 102, wherein only the second to last, third to last, and fourth to last nucleotides in the 3' terminal region are modified.

[0971] 106. The guide RNA of any one of embodiments 79 to 100, further comprising a PS bond between the first three, four or five nucleotides at the 5' end of the 5' terminus.

[0972] 107. The guide RNA of embodiment 106, wherein the first four nucleotides at the 5' end of the 5' terminus are linked to a PS bond.

[0973] 108. The guide RNA of any one of embodiments 79 to 100, further comprising a PS bond between the last three, four or five nucleotides at the 3' end of the 3' terminus.

[0974] 109. The guide RNA of embodiment 108, wherein the last four nucleotides at the 3' end of the 3' terminus are linked to a PS bond.

[0975] 110. The guide RNA of any one of embodiments 79 to 100, wherein the first three or four nucleotides at the 5' end of the 5' terminus and the last three or four nucleotides at the 3' end of the 3' terminus comprise a 2'-O-Me modification.

[0976] 111. The guide RNA of any one of embodiments 79 to 100, wherein the first three or four nucleotides at the 5' end of the 5' end are linked to a PS bond, and the last three or four nucleotides at the 3' end of the 3' end are linked to a PS bond.

[0977] 112. The guide RNA of any one of embodiments 79 to 100, wherein the first three or four nucleotides at the 5' end of the 5' end comprise a 2'-O-Me modification and are linked to a PS bond, and the last three or four nucleotides at the 3' end of the 3' end comprise a 2'-O-Me modification and are linked to a PS bond.

[0978] 113. The guide RNA of any one of embodiments 79 to 100, wherein at least two of nucleotides LS8, LS9, LS10, LS11 and LS12 comprise a 2'-O-Me modification.

[0979] 114. The guide RNA of any one of embodiments 79 to 100, wherein at least LS8 and LS10 are modified with 2'-O-Me.

[0980] 115. The guide RNA of any one of embodiments 79 to 100, wherein at least 50% of the nucleotides in the linker region are modified with 2'-O-Me.

[0981] 116. The guide RNA of embodiment 115, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the linker region are modified with 2'-O-Me.

[0982] 117. The guide RNA of embodiment 115, wherein at least N2 to N6 are modified with 2'-O-Me.

[0983] 118. The guide RNA of any one of embodiments 79 to 100, wherein at least ten of the linker region nucleotides are modified with 2'-O-Me.

[0984] 119. The guide RNA of embodiment 115, wherein at least N2 to N6 are modified with 2'-O-Me.

[0985] 120. The guide RNA of any one of embodiments 79 to 100, wherein at least 50% of the nucleotides in the protuberance region are modified with 2'-O-Me.

[0986] 121. The guide RNA of embodiment 120, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the protuberance region are modified with 2'-O-Me.

[0987] 122. The guide RNA of embodiment 121, wherein B2, B3 and / or B4 are modified with 2'-O-Me.

[0988] 123. The guide RNA of any one of embodiments 79 to 100, wherein at least three of the nucleotides in the protuberance region are modified with 2'-O-Me.

[0989] 124. The guide RNA of embodiment 123, wherein B2, B3 and / or B4 are modified with 2'-O-Me.

[0990] 125. The guide RNA of any one of embodiments 79 to 124, wherein the guide RNA is a sgRNA.

[0991] 126. The guide RNA of any one of embodiments 79 to 125, wherein the guide RNA is a dgRNA.

[0992] 127. The guide RNA of embodiment 81 or 82, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the hairpin 1 region and the hairpin 2 region are modified with 2'-OMe.

[0993] 128. The guide RNA of any one of embodiments 79 to 127, wherein the sgRNA forms a ribonucleoprotein complex with Streptococcus pyogenes Cas9.

[0994] 129. A single guide RNA (sgRNA) comprising or consisting of a 2'-O-Me modified nucleotide at:

[0995] a. The first three nucleotides at the 5' end of the 5' terminus;

[0996] b. Each nucleotide in the upper stem region;

[0997] c. Each nucleotide in the hairpin 1 region;

[0998] d. Nucleotides between hairpin 1 and hairpin 2;

[0999] e. each nucleotide in region 2 of the hairpin; and

[1000] f. The last four nucleotides at the 3' end of the 3' terminus.

[1001] 130. The sgRNA of embodiment 129, further comprising a PS bond connecting the first four nucleotides at the 5' end of the 5' end and a PS bond connecting the last four nucleotides at the 3' end of the 3' end.

[1002] 131. An sgRNA having a modification pattern as shown in SEQ ID NO: 350, 351, 352 or 353, as shown in Table 4.

[1003] 132. A single guide RNA (sgRNA) comprising or consisting of a 2'-O-Me modified nucleotide at:

[1004] a. The first three, four, five or seven nucleotides at the 5' end of the 5' terminus;

[1005] b. Each nucleotide in the upper stem region;

[1006] c. Each nucleotide in the hairpin 1 region;

[1007] d. Nucleotides between hairpin 1 and hairpin 2;

[1008] e. each nucleotide in region 2 of the hairpin; and

[1009] f. The last four nucleotides at the 3' end of the 3' terminus.

[1010] 133. A single guide RNA (sgRNA) comprising or consisting of a 2'-O-Me modified nucleotide at:

[1011] a. The first three, four, five or seven nucleotides at the 5' end of the 5' terminus;

[1012] b. each of the nucleotides US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11 and US12;

[1013] c. each of nucleotides H1-1, H1-2, H1-3, H1-4, H1-5, H1-6, H1-7, H1-8, H1-9, H1-10, H1-11, and H1-12;

[1014] d. Nucleotides between hairpin 1 and hairpin 2;

[1015] e. each of nucleotides H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-8, H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15; and

[1016] f. The last four nucleotides at the 3' end of the 3' terminus.

[1017] 134. The sgRNA of any one of embodiments 129 to 133, further comprising a PS bond connecting the first four nucleotides at the 5' end of the 5' end and a PS bond connecting the last four nucleotides at the 3' end of the 3' end.

[1018] 135. An sgRNA having a modification pattern of SEQ ID NO: 350, 351, 352 or 353 as shown in Table 4.

[1019] 136. The sgRNA of any one of embodiments 129 to 135, wherein the sgRNA forms a ribonucleoprotein complex with Streptococcus pyogenes Cas9.

[1020] 137. a crispr RNA (crRNA) comprising one or more modifications in one or more of the following regions:

[1021] a. The first five nucleotides at the 5' end of the 5' terminus;

[1022] b. Lower stem region;

[1023] c. protrusion area;

[1024] d. upper stem region; and

[1025] e. The last five nucleotides at the 3' end of crRNA.

[1026] 138. The crRNA of embodiment 136, further comprising a 5' end modification, wherein the 5' end modification comprises one or more phosphorothioate bonds within the first seven nucleotides at the 5' end of the 5' terminus.

[1027] 139. The crRNA of embodiment 136, further comprising a 5' end modification, wherein the 5' end modification comprises at least two phosphorothioate bonds within the first seven nucleotides at the 5' end of the 5' terminus.

[1028] 140. The crRNA of any one of embodiments 137 to 139, wherein at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[1029] 141. The crRNA of any one of embodiments 137 to 140, wherein at least one modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[1030] 142. The crRNA of any one of embodiments 137 to 141, wherein at least one modification comprises a phosphorothioate (PS) bond between nucleotides.

[1031] 143. The crRNA of any one of embodiments 137 to 142, wherein the first three nucleotides at the 5' end of the 5' end and the last three nucleotides at the 3' end of the 3' end are modified.

[1032] 144. The crRNA of any one of embodiments 137 to 143, wherein the first four nucleotides at the 5' end of the 5' end and the last four nucleotides at the 3' end of the 3' end are linked to phosphorothioate (PS) bonds.

[1033] 145. The crRNA of embodiment 143, wherein the modification comprises 2'-O-Me.

[1034] 146. The crRNA of embodiment 143, wherein the modification comprises 2'-F.

[1035] 147. The crRNA of any one of embodiments 137 to 146, wherein the first four nucleotides at the 5' end of the 5' end and the last four nucleotides at the 3' end of the 3' end are linked to a PS bond. And wherein the first three nucleotides at the 5' end of the 5' end and the last three nucleotides at the 3' end of the 3' end comprise a 2'-O-Me modification.

[1036] 148. The crRNA of any one of embodiments 137 to 146, wherein the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus are linked to a PS bond, and wherein the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus comprise a 2'-F modification.

[1037] 149. The crRNA of any one of embodiments 137 to 148, wherein LS1 and LS6 are modified with 2'-O-Me.

[1038] 150. The crRNA of any one of embodiments 137 to 149, wherein each of the upper stem region nucleotides is modified with 2'-O-Me.

[1039] 151.crispr RNA (crRNA) comprising 2'-O-Me modified nucleotides at:

[1040] a. LS1 and LS6 in the lower stem region; and

[1041] b. Each nucleotide in the upper stem region.

[1042] 152. The crRNA of embodiment 151, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[1043] 153. The crRNA of embodiment 151 or embodiment 152, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end of the 5' end and 2'-O-Me or 2'-F modified nucleotides at the last three nucleotides at the 3' end of the 3' end.

[1044] 154. The crRNA of any one of embodiments 137 to 153, wherein LS1, LS2 and LS6 are modified with 2'-F.

[1045] 155. The crRNA of any one of embodiments 137 to 154, wherein each nucleotide in the protrusion region is modified with 2'-F.

[1046] 156. a crispr RNA (crRNA) comprising a 2'-F modified nucleotide at:

[1047] a. LS1, LS2 and LS6 in the lower stem region; and

[1048] b. Each nucleotide in the bulge region.

[1049] 157. The crRNA of any one of embodiments 137 to 156, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[1050] 158. The crRNA of any one of embodiments 137 to 157, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end of the 5' end and 2'-O-Me or 2'-F modified nucleotides at the last three nucleotides at the 3' end of the 3' end.

[1051] 159. A crRNA comprising any one of SEQ ID Nos: 1 to 187, comprising the modification of Table 4.

[1052] 160. A crRNA comprising any one of SEQ ID Nos: 19 to 31, 53 to 73, 104 to 130 and 161 to 187, comprising the modification of Table 4.

[1053] 161. A crRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity to any one of SEQ ID Nos: 19 to 31, 53 to 73, 104 to 130 and 161 to 187, wherein the modification at each nucleotide of the crRNA corresponding to the nucleotide of the reference sequence identifier in Table 4 is the same or equivalent to the modification shown in the reference sequence identifier in Table 4.

[1054] 162. The crRNA of any one of embodiments 159 to 161, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[1055] 163. The crRNA of any one of embodiments 137 to 162, wherein the crRNA combined with the trRNA forms a ribonucleoprotein complex with Streptococcus pyogenes Cas9.

[1056] 164. tracrRNA (trRNA) comprising one or more modifications in one or more of the following regions:

[1057] a. The first five nucleotides at the 5' end of the 5' terminus;

[1058] b. Upper stem region;

[1059] c. protrusion area;

[1060] d. Lower stem region;

[1061] e. Connection area;

[1062] f. Hairpin 1 area;

[1063] g. Hairpin 2 area; and

[1064] h. The last five nucleotides at the 3' end of the 3' terminus.

[1065] 165. The trRNA of embodiment 164, wherein at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[1066] 166. The trRNA of embodiment 164 or embodiment 165, wherein at least one modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[1067] 167. The trRNA of any one of embodiments 164 to 166, wherein at least one modification comprises a phosphorothioate (PS) bond between nucleotides.

[1068] 168. The trRNA of any one of embodiments 164 to 167, wherein the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus are linked to phosphorothioate (PS) bonds.

[1069] 169. The trRNA of any one of embodiments 164 to 168, wherein the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus are modified.

[1070] 170. The trRNA of embodiment 169, wherein the modification comprises 2'-O-Me.

[1071] 171. The trRNA of embodiment 169, wherein the modification comprises 2'-F.

[1072] 172. The trRNA of any one of embodiments 164 to 171, wherein the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus are linked to a PS bond, and wherein the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus comprise a 2'-O-Me modification.

[1073] 173. The trRNA of any one of embodiments 164 to 171, wherein the first four nucleotides at the 5' end of the 5' terminus and the last four nucleotides at the 3' end of the 3' terminus are linked to a PS bond, and wherein the first three nucleotides at the 5' end of the 5' terminus and the last three nucleotides at the 3' end of the 3' terminus comprise a 2'-F modification.

[1074] 174. The trRNA of any one of embodiments 164 to 173, wherein each nucleotide in the upper stem region is modified with 2'-O-Me.

[1075] 175. The trRNA of any one of embodiments 164 to 174, wherein B1 and B2 within the protuberance region are modified with 2′-O-Me.

[1076] 176. The trRNA of any one of embodiments 164 to 175, wherein N3, N4, N5, N15, N16, N17 and / or N18 in the linker region are modified with 2'-O-Me.

[1077] 177. The trRNA of any one of embodiments 164 to 176, wherein each nucleotide in the hairpin 1 region is modified with 2'-O-Me.

[1078] 178. The trRNA of any one of embodiments 164 to 177, wherein each nucleotide in the hairpin 2 region is modified with 2'-O-Me.

[1079] 179. tracrRNA (trRNA) comprising 2'-O-Me modified nucleotides at:

[1080] a. Each nucleotide in the upper stem region;

[1081] b. B1 and / or B2 in the protrusion area;

[1082] c. N3, N4, N5, N15, N16, N17 and / or N18 in the connection region;

[1083] d. each nucleotide in the hairpin 1 region; and

[1084] e. Each nucleotide in the hairpin 2 region.

[1085] 180. The trRNA of embodiment 179, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[1086] 181. The trRNA of embodiment 179 or embodiment 180, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end of the 5' end and 2'-O-Me or 2'-F modified nucleic acid at the last three nucleotides at the 3' end of the 3' end.

[1087] 182. The trRNA of any one of embodiments 164 to 181, wherein N15, N16, N17 and N18 are modified with 2'-F.

[1088] 183. The trRNA of any one of embodiments 164 to 182, wherein LS1, LS3 and LS5 are modified with 2'-F, and LS2, LS4 and LS6 are modified with 2'-O-Me.

[1089] 184. The trRNA of any one of embodiments 164 to 183, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[1090] 185. The trRNA of any one of embodiments 164 to 184, further comprising 2'-O-Me or 2'-F modified nucleotides at the first three nucleotides at the 5' end of the 5' end and 2'-O-Me or 2'-F modified nucleotides at the last three nucleotides at the 3' end of the 3' end.

[1091] 186. A trRNA comprising any one of SEQ ID Nos: 188 to 227, comprising the modification of Table 4.

[1092] 187. trRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 or 70% identity to any one of SEQ ID Nos: 188 to 227, wherein the modification at each nucleotide of the trRNA corresponding to the nucleotide of the reference sequence identifier in Table 4 is the same or equivalent to the modification shown in the reference sequence identifier in Table 4.

[1093] 188. The trRNA of any one of embodiments 186 to 187, further comprising three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end of the 5' end and three PS bonds connecting the last four nucleotides at the 3' end of the 3' end.

[1094] 189. The trRNA of any one of embodiments 164 to 188, wherein the trRNA combined with the crRNA forms a ribonucleoprotein complex with Streptococcus pyogenes Cas9.

[1095] 190. A dual guide comprising crRNA and trRNA, wherein the crRNA comprises any one of SEQ ID Nos: 1 to 187 as shown in Table 4, and wherein the trRNA comprises a nucleic acid of any one of SEQ ID Nos: 188 to 227 as shown in Table 4.

[1096] 191. A dual guide comprising the crRNA of any one of embodiments 137 to 163 and the trRNA of any one of embodiments 164 to 189.

[1097] 192. A dual guide comprising the crRNA of any one of embodiments 137 to 163 and an unmodified trRNA.

[1098] 193. A dual guide comprising an unmodified crRNA and a trRNA as in any one of embodiments 137 to 189.

[1099] 194. The dual guide of any one of embodiments 190 to 193, wherein the dual guide forms a ribonucleoprotein complex with Streptococcus pyogenes Cas9.

[1100] 195. A LNP composition comprising the sgRNA of any one of embodiments 1 to 78 and 129 to 136.

[1101] 196. An LNP composition comprising a gRNA, wherein the gRNA comprises or consists of an RNA as described in any one of embodiments 1 to 193.

[1102] 197. A composition comprising the sgRNA of any one of embodiments 1 to 78 and 129 to 136 associated with a lipid nanoparticle (LNP).

[1103] 198. A composition comprising the crRNA of any one of embodiments 137 to 163 associated with a lipid nanoparticle (LNP).

[1104] 199. A composition comprising the trRNA of any one of embodiments 164 to 189 associated with a lipid nanoparticle (LNP).

[1105] 200. A composition comprising an sgRNA as described in any one of embodiments 1 to 78 and 129 to 136, a gRNA as described in any one of embodiments 79 to 128, a crRNA as described in any one of embodiments 137 to 163, a trRNA as described in any one of embodiments 164 to 189, a dgRNA as described in any one of embodiments 190 to 194, or a composition as described in any one of embodiments 195 to 199, further comprising a nuclease or an mRNA encoding a nuclease.

[1106] 201. The composition of embodiment 200, wherein the nuclease is a Cas protein.

[1107] 202. The composition of embodiment 201, wherein the Cas protein is Cas9.

[1108] 203. The composition of embodiment 202, wherein Cas9 is Streptococcus pyogenes Cas9.

[1109] 204. The composition of any one of embodiments 200 to 203, wherein the nuclease is a nickase.

[1110] 205. The composition of any one of embodiments 200 to 204, wherein the nuclease is a modified nuclease.

[1111] 206. The composition of embodiment 205, wherein the modified nuclease comprises a nuclear localization signal (NLS).

[1112] 207. A composition as described in any one of embodiments 200 to 206, which comprises mRNA encoding a nuclease.

[1113] 208. A pharmaceutical preparation comprising an sgRNA as described in any one of embodiments 1 to 78 and 129 to 136, a gRNA as described in any one of embodiments 79 to 128, a crRNA as described in any one of embodiments 137 to 163, a trRNA as described in any one of embodiments 164 to 189, a dgRNA as described in any one of embodiments 190 to 194, or a composition as described in any one of embodiments 195 to 207, and a pharmaceutically acceptable carrier.

[1114] 209. A method for modifying a target DNA, comprising delivering to a cell a Cas protein or a nucleic acid encoding a Cas protein and any one or more of the following:

[1115] a. The sgRNA of any one of embodiments 1 to 78 and 129 to 136;

[1116] b. The gRNA of any one of embodiments 79 to 128;

[1117] c. a crRNA according to any one of embodiments 137 to 163;

[1118] d. The trRNA of any one of embodiments 164 to 189;

[1119] e. The dgRNA of any one of embodiments 190 to 194;

[1120] f. a composition according to any one of embodiments 195 to 207; or

[1121] g. A pharmaceutical preparation according to embodiment 208.

[1122] 210. The method of embodiment 209, wherein the method results in an insertion or deletion in the gene.

[1123] 211. The method of embodiment 209, further comprising delivering the template to the cell, wherein at least a portion of the template is incorporated into the target DNA at or near the site of the double-strand break induced by the Cas protein.

[1124] 212. The sgRNA of any one of embodiments 1 to 78 and 129 to 136, for use in the preparation of a medicament for treating a disease or disorder.

[1125] 213. The crRNA of any one of embodiments 737 to 163, for use in the preparation of a medicament for treating a disease or disorder.

[1126] 214. The trRNA of any one of embodiments 164 to 189, for use in the preparation of a medicament for treating a disease or disorder.

[1127] 215. A combination of a crRNA as described in any one of embodiments 137 to 163 and a trRNA as described in any one of embodiments 164 to 189, for use in the preparation of a medicament for treating a disease or disorder.

[1128] 216. A pharmaceutical formulation according to embodiment 208, for use in the preparation of a medicament for treating a disease or condition.

Claims

1. A single guide RNA comprising a 5' end modification and one or more of the following modifications: a. Upper stem area; b. Hairpin 1 area; and c. Hairpin 2 area, wherein the 5' end modification comprises at least two phosphorothioate (PS) bonds within the first seven nucleotides at the 5' end of the 5' terminus.

2. The sgRNA of claim 1, wherein at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

3. The sgRNA of claim 1 or claim 2, wherein at least one modification comprises a 2'-fluoro (2'-F) modified nucleotide.

4. The sgRNA of any one of claims 1 to 3, wherein at least one modification comprises a phosphorothioate (PS) bond between nucleotides.

5. The sgRNA of any one of claims 1 to 4, wherein the sgRNA comprises one or more modifications in the upper stem region.

6. The sgRNA of claim 5, comprising modifications at US1 to US12.

7. The sgRNA of any one of claims 1 to 6, wherein the sgRNA comprises one or more modifications in the hairpin 1 region.

8. The sgRNA of claim 7, wherein the sgRNA comprises a modification at H1-1.

9. The sgRNA of any one of claims 1 to 8, wherein the sgRNA comprises one or more modifications in the hairpin 2 region.

10. The sgRNA of claim 9, wherein the sgRNA comprises a modification at H2-1.

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