Modified guide RNA

Abstract

The present disclosure relates to modified single-guide and dual-guide RNAs with improved in vitro and in vivo activity in gene editing methods. [Selection diagram] None

Description

Technical Field

[0001] This application includes a Sequence Listing that has been electronically submitted in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy was created on December 7, 2017, has the name 01155-0004-00PCT_SeqList.txt, and is 118,877 bytes in size.

[0002] Cross-Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 431,756, filed on December 8, 2016, which is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to the field of gene editing using the CRISPR / Cas system, which is part of a prokaryotic immune system that recognizes and cleaves exogenous genetic elements. The CRISPR / Cas system relies on a single nuclease called CRISPR-associated protein 9 (Cas9) to induce site-specific cleavage of DNA. Cas9 is guided to a specific DNA sequence by a short-chain RNA molecule called guide RNA (gRNA). The guide RNA includes a trRNA (also known as tracrRNA) and a crisprRNA (crRNA). The trRNA and crRNA may be contained within a single guide RNA (sgRNA), or may be contained within two separate RNA molecules of a dual guide RNA (dgRNA). Cas9 in combination with the trRNA and crRNA or sgRNA is referred to as a Cas9 ribonucleoprotein complex (RNP).

[0004] Oligonucleotides, particularly RNA, may sometimes be degraded in cells and in serum by endonuclease cleavage or exonuclease cleavage. Improved methods and compositions for preventing such degradation, improving the stability of the gRNA, and enhancing gene editing efficiency are desired, particularly for therapeutic applications.

Summary of the Invention

Means for Solving the Problem

[0005] In some embodiments, a therapeutic genome editing tool comprising a modified guide RNA is provided. The modified guide RNAs 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) that cleaves target DNA. In some embodiments, the guide RNA is a sgRNA. In some embodiments, the guide RNA is a dgRNA. In some embodiments, the guide RNA is a tracrRNA. In some embodiments, the guide RNA is a crRNA.

[0006] The guide RNAs described herein comprise at least one modified nucleotide. Modifications may include 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), 2'-fluoro (2'-F), phosphorothioate (PS) linkages between nucleotides, G-C substitutions, and inverted deoxyribonucleotide linkages between nucleotides and their equivalents. Embodiments of the invention include the following.

[0007] In some embodiments, a single guide RNA (sgRNA) is included with one or more modifications in one or more of a 5'-end modification and the upper stem region; the hairpin 1 region; and the hairpin 2 region, and the 5'-end modification comprises at least two phosphorothioate linkages among the first 7 nucleotides at the 5'-terminus of the 5'-end. 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.

[0008] In some embodiments, the sgRNA comprises modifications in US1-US12, and / or in H1-1, and / or in H2-1. In some embodiments, the sgRNA comprises modifications in H1-1 - H1-12 and / or H2-1 - H2-15. In some embodiments, the sgRNA comprises one or more modifications in 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.

[0009] In some embodiments, the sgRNA comprises modifications at the 5' end and / or at the 3' end. In some embodiments, the sgRNA comprises 3' terminal modifications within the 3' end. In some embodiments, the sgRNA comprises modifications in at least two of the last four nucleotides at the 3' terminus of the 3' end. In some embodiments, the sgRNA comprises 5' terminal modifications within the 5' end. In some embodiments, the sgRNA comprises modifications in at least two of the first four nucleotides at the 5' terminus of the 5' end. In some embodiments, the sgRNA comprises 3' terminal modifications within the 3' end and 5' terminal modifications within the 5' end. In some embodiments, the sgRNA comprises modifications in at least two of the last four nucleotides at the 3' terminus of the 3' end and in at least two of the first four nucleotides at the 5' terminus of the 5' end. In some cases, these modifications are 2'-O-Me, 2'-F, 2'-O-moe, or phosphorothioate (PS) linkages that link nucleotides. In some embodiments, the sgRNA comprises PS linkages between at least two of the last four nucleotides at the 3' terminus of the 3' end and / or at least two of the first four nucleotides at the 5' terminus of the 5' end. In some cases, the sgRNA comprises 5' and 3' ends with more than one modification described herein, such as PS linkages and 2'-O-Me modifications.

[0010] In some embodiments, the sgRNA contains modifications in the bulge region. In some embodiments, 50% of the nucleotides in the bulge region are modified, and the modification is 2'-O-Me or 2'-F.

[0011] In some embodiments, the sgRNA contains modifications in the nexus region. In some embodiments, the sgRNA contains modifications at N15, N16, N17, and / or N18 in the nexus region, and the modification is 2'-O-Me or 2'-F. In some cases, N16, N17, and N18 are linked by a PS bond.

[0012] In some embodiments, the sgRNA contains at least the first 3 nucleotides at the 5' end of the modified 5' end and the last 3 nucleotides at the 3' end of the 3' end.

[0013] In some embodiments, the sgRNA contains modifications at the 3' end and / or 5' end. In some cases, the first 4 nucleotides at the 5' end of the 5' end and the last 4 nucleotides at the 3' end of the 3' end are linked by phosphorothioate (PS) bonds. In some embodiments, the 5' and 3' modifications include 2'-O-Me or 2'-O-moe. In some embodiments, the 5' and 3' modifications include 2'-F. In some embodiments, the 5' and / or 3' modifications include PS bonds that link nucleotides. In some embodiments, the 5' and / or 3' modifications include one or more of 2'-O-Me, 2'-O-moe, 2'-F, and PS bonds that link nucleotides.

[0014] In some embodiments, the sgRNA comprises modifications at the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus. In some cases, these modifications are PS linkages that link (i.e., PS linkages that link the first 4 nucleotides and the last 4 nucleotides). In some embodiments, the sgRNA further comprises 2'-O-Me modifications at the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus.

[0015] In some embodiments, the sgRNA comprises modifications at the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus, the modifications being at least PS linkages that link the 4 nucleotides, and further, the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus comprise 2'-O-Me, 2'-O-moe, or 2'-F modifications.

[0016] In some embodiments, the sgRNA comprises modifications LS1, LS6, LS7, LS8, LS11, and LS12, the modifications being 2'-O-Me or 2'-F.

[0017] In some embodiments, the sgRNA comprises modifications at each of the nucleotides in the bulge region, the modifications being 2'-O-Me or 2'-F.

[0018] In some embodiments, the sgRNA comprises modifications at each of the nucleotides in the upper stem region, the modifications being 2'-O-Me or 2'-F.

[0019] In some embodiments, the sgRNA comprises modifications at each of the nucleotides in the hairpin 1 region, the modifications being 2'-O-Me or 2'-F.

[0020] In some embodiments, the sgRNA comprises a modification at each nucleotide in the hairpin 2 region, and the modification is 2'-O-Me or 2'-F.

[0021] In some embodiments, the following positions: a. the first 3 nucleotides at the 5' end of the 5' terminus, and b. LS1, LS6, LS7, LS8, LS11, and / or LS12 in the lower stem region, and c. B1 and / or B2 in the bulge region, and d. each nucleotide in the upper stem region, and e. N16, N17, and / or N18 in the nexus region, and f. each nucleotide in the hairpin 1 region, and g. each nucleotide in the hairpin 2 region, and h. the last 4 nucleotides at the 3' end of the 3' terminus, include an sgRNA comprising 2'-O-Me modified nucleotides. In some embodiments, B3 - B6 are modified with 2'-O-Me. In some cases, the sgRNA further comprises 3 phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end of the 5' terminus and 3 PS bonds connecting the last 4 nucleotides at the 3' end of the 3' terminus. In some embodiments, the sgRNA comprises a 2'-F modification at LS9 and LS10. In some embodiments, the sgRNA comprises a 2'-F modification at N15, N16, N17, and N18. In some embodiments, the sgRNA comprises a 2'-F modification at H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15. In some embodiments, the sgRNA comprises a 2'-F modification at the second last, third last, and fourth last nucleotides at the 3' end of the 3' terminus.

[0022] In some embodiments, the following positions: a. LS9 and LS10 in the lower stem region, and b. N15, N16, N17, and N18 in the Nexus region, and c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 in the Hairpin 2 region, wherein the sgRNA contains 2'-F modified nucleotides. In some embodiments, the sgRNA contains 2'-F modified nucleotides at the second last, third last, and fourth last nucleotides at the 3' end. In some embodiments, the sgRNA contains three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' end of the 3' terminus. In some embodiments, the sgRNA contains 2'-O-Me or 2'-F modified nucleotides at the first 3 nucleotides at the 5' end of the 5' terminus and also contains 2'-O-Me or 2'-F modified nucleotides at three of the last 4 nucleotides at the 3' end of the 3' terminus.

[0023] In some embodiments, a. 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end of the 5' terminus, and b. Optional 2'-O-Me modified nucleotides in LS1 and / or LS6, and c. 2'-O-Me modified nucleotides in US1-US12, and d. 2'-O-Me modified nucleotides in H1-1 - H1-12 e. Optional 2'-O-Me modified nucleotides between Hairpin 1 and Hairpin 2, and f. 2'-O-Me modified nucleotides in H2-1 - H2-15, and g. 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end of the 3' terminus, and optionally, The sgRNA further includes three phosphorothioate (PS) linkages that link the first four nucleotides at the 5'-end of the 5'-terminus and three PS linkages that link the last four nucleotides at the 3'-end of the 3'-terminus.

[0024] In some embodiments, a. 2'-O-Me modified nucleotides in the first 3 nucleotides at the 5'-end of the 5'-terminus, and b. 2'-F modified nucleotides in LS1-LS6, and c. 2'-O-Me modified nucleotides in US1-US12, and d. 2'-O-Me modified nucleotides in H1-1-H1-12, and e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and f. 2'-O-Me modified nucleotides in H2-1-H2-15, and g. 2'-O-Me modified nucleotides in the last four nucleotides at the 3'-end of the 3'-terminus, and optionally, the sgRNA further includes three phosphorothioate (PS) linkages that link the first four nucleotides at the 5'-end of the 5'-terminus and three PS linkages that link the last four nucleotides at the 3'-end of the 3'-terminus.

[0025] In some embodiments, a. 2'-O-Me modified nucleotides in the first three nucleotides at the 5'-terminus, and b. 2'-F modified nucleotides in LS2-LS5, and c. 2'-O-Me modified nucleotides in LS1 and LS6, and d. 2'-O-Me modified nucleotides in US1-US12, and e. 2'-O-Me modified nucleotides in H1-1-H1-12, and f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and 2'-O-Me modified nucleotides in g.H2-1 to H2-15, and h.2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, and optionally, Three phosphorothioate (PS) linkages connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS linkages connecting the last 4 nucleotides at the 3' end of the 3' terminus are further included, and the sgRNA is included.

[0026] In some embodiments, a.2'-O-Me modified nucleotides in the first 3 nucleotides at the 5' terminus, and b.2'-O-Me modified nucleotides in US1 to US12, and c.2'-O-Me modified nucleotides in LS7, LS8, LS11, and LS12, and d.2'-O-Me modified nucleotides in H1-1 to H1-12, and e.2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and f.2'-O-Me modified nucleotides in H2-1 to H2-15, and g.2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, and and optionally, three phosphorothioate (PS) linkages connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS linkages connecting the last 4 nucleotides at the 3' end of the 3' terminus are further included, and the sgRNA is included.

[0027] In some embodiments, a.2'-O-Me modified nucleotides in the first 3 nucleotides at the 5' terminus, and b.2'-O-Me modified nucleotides in US1 to US12, and c.2'-O-Me modified nucleotides in LS7, LS8, LS11, and LS12, and d. 2'-F modified nucleotides in LS9 and LS10, and e. 2'-O-Me modified nucleotides in H1-1 to H1-12, and f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and g. 2'-O-Me modified nucleotides in H2-1 to H2-15, and h. 2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, and includes optionally, three phosphorothioate (PS) bonds that link the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds that link the last 4 nucleotides at the 3' end of the 3' terminus, and further includes a sgRNA.

[0028] In some embodiments, a. 2'-O-Me modified nucleotides in the first 3 nucleotides at the 5' terminus, and b. 2'-O-Me modified nucleotides in US1 to US12, and c. 2'-O-Me modified nucleotides in LS8, LS10, and LS12, and d. 2'-O-F modified nucleotides in LS7, LS9, and LS11, and e. 2'-O-Me modified nucleotides in H1-1 to H1-12, and f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and g. 2'-O-Me modified nucleotides in H2-1 to H2-15, and h. 2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, and includes, and optionally three phosphorothioate (PS) bonds that link the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds that link the last 4 nucleotides at the 3' end of the 3' terminus, and further includes a sgRNA.

[0029] In some embodiments, a. 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, and b. 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12, and c. 2'-O-Me modified nucleotides in US1-US12, and d. 2'-O-Me modified nucleotides in H1-1 - H1-12, and e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and f. 2'-O-Me modified nucleotides in H2-1 - H2-15, and g. 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end, and optionally, Three phosphorothioate (PS) linkages connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS linkages connecting the last 4 nucleotides at the 3' end of the 3' terminus are further included, and the sgRNA is included.

[0030] In some embodiments, a. 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, and b. 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12, and c. 2'-F modified nucleotides in LS9 and LS10, and d. 2'-O-Me modified nucleotides in US1-US12, and e. 2'-O-Me modified nucleotides in H1-1 - H1-12, and f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and g. 2'-O-Me modified nucleotides in H2-1 - H2-15, and h. 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end, and optionally, The sgRNA further includes three phosphorothioate (PS) linkages that link the first four nucleotides at the 5'-end of the 5'-terminus and three PS linkages that link the last four nucleotides at the 3'-end of the 3'-terminus.

[0031] In some embodiments, a. 2'-O-Me modified nucleotides in the first three nucleotides at the 5'-end of the 5'-terminus, and b. 2'-O-Me modified nucleotides in US1-US12, and c. 2'-O-Me modified nucleotides in H1-1 - H1-12, and d. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and e. 2'-O-Me modified nucleotides in H2-1 - H2-8, and f. 2'-F modified nucleotides in H2-9 - H2-15, and g. 2'-F modified nucleotides in the second last, third last, and fourth last nucleotides at the 3'-terminus, and h. 2'-O-Me modified nucleotides in the last nucleotide at the 3'-terminus, and optionally, The sgRNA further includes three phosphorothioate (PS) linkages that link the first four nucleotides at the 5'-end of the 5'-terminus and three PS linkages that link the last four nucleotides at the 3'-end of the 3'-terminus.

[0032] In some embodiments, a. 2'-O-Me modified nucleotides in the first three nucleotides at the 5'-end of the 5'-terminus, and b. 2'-O-Me modified nucleotides in US1-US12, and c. 2'-O-Me modified nucleotides in H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12; and d. 2'-F modified nucleotides in H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11, and e. the 2'-F modified nucleotides between hairpin 1 and hairpin 2, and f. the 2'-F modified nucleotides in H2-2, H2-4, H2-6, H2-8, H2-10, H2-12; and H2-14; and g. the 2'-O-Me modified nucleotides in H2-1, H2-3, H2-5, H2-7, H2-9, H2-11; H2-13, and H2-15; and h. the 2'-F modified nucleotides in the second last and fourth last nucleotides at the 3'-terminus, and i. the 2'-O-Me modified nucleotides in the third last and last nucleotides at the 3'-terminus of the 3'-end, and includes and optionally, further includes three phosphorothioate (PS) bonds that link the first four nucleotides at the 5'-terminus of the 5'-end and three PS bonds that link the last four nucleotides at the 3'-terminus of the 3'-end, and the sgRNA is included.

[0033] In some embodiments, a. the 2'-O-Me modified 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 b. the 2'-F modified nucleotides in 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; and includes optionally, three phosphorothioate (PS) bonds that link the first four nucleotides at the 5'-terminus of the 5'-end and three PS bonds that link the last four nucleotides at the 3'-terminus of the 3'-end, and optionally, c. the 2'-O-Me modified nucleotides in the last and third last nucleotides at the 3'-terminus of the 3'-end; and / or d. The sgRNA further includes 2'-F modified nucleotides at the second last, fourth last, and / or last nucleotides at the 3' end of the 3' terminus.

[0034] In some embodiments, the sgRNA includes any nucleic acid of SEQ ID NOs: 228 - 353 containing the modifications in Table 4. In some embodiments, the sgRNA includes any of SEQ ID NOs: 228 - 332 containing the modifications in Table 4. In some embodiments, the sgRNA includes any of SEQ ID NOs: 235 - 240, 265 - 285, and 309 - 329 containing the modifications in Table 4. In some embodiments, the sgRNA includes SEQ ID NO: 240. In some embodiments, the sgRNA includes SEQ ID NO: 240 containing the modifications in Table 4. In some embodiments, the sgRNA includes SEQ ID NO: 242. In some embodiments, the sgRNA includes SEQ ID NO: 358. In additional embodiments, the sgRNA includes 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 the nucleic acids of SEQ ID NOs: 235 - 240, 265 - 285, and 309 - 329, and the modification at each nucleotide of the sgRNA corresponding to the nucleotide of the reference sequence identifier in Table 4 is the same as or equivalent to the modification shown in the reference sequence identifier in Table 4. Optionally, the sgRNA further includes three phosphorothioate (PS) bonds linking the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds linking the last 4 nucleotides at the 3' end of the 3' terminus. In some embodiments, the sgRNA further includes at least three PS bonds linking the nucleotides in the hairpin 1 region. In some embodiments, the sgRNA further includes at least three PS bonds linking the nucleotides in the hairpin 2 region. In some embodiments, the sgRNA further includes at least three PS bonds linking the nucleotides in the upper stem region. In some embodiments, the sgRNA forms a ribonucleoprotein complex with Cas9 of S. Pyogenes.

Brief Description of the Drawings

[0035]

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Mode for Carrying Out the Invention

[0036] Modified guide RNAs, including dual guide RNAs and single guide RNAs for use in gene editing methods, are provided herein. The modified guides are more stable compared to their unmodified counterparts and exhibit improved in vitro and in vivo efficacy. The sequences of the guide RNAs that were engineered and tested are shown in Table 4.

Table 1

[0037] "Guide RNA" and "gRNA" are used interchangeably herein to generically refer to either single guide RNA (sgRNA), trans-activating crRNA (trRNA, also known as tracrRNA), or CRISPR RNA (crRNA). crRNA and trRNA may associate on one RNA molecule (single guide RNA [sgRNA]) or may be in two separate RNA molecules (dual guide RNA [dgRNA]). "Guide RNA" or "gRNA" refers to each type.

[0038] The trRNA sequence may be naturally occurring or may contain modifications or variations compared to a naturally occurring sequence.

[0039] As used herein, "editing efficiency" or "editing percentage" or "editing percent" is the total number of sequence reads having nucleotide insertions or deletions in the target region of interest relative to the total number of sequence reads after cleavage by Cas RNP.

[0040] As used herein, "hairpin" refers to a loop of nucleic acid created when a nucleic acid strand folds and forms base pairs with another segment of the same strand. A hairpin may form a structure that includes a loop or a U-shape. In some embodiments, the hairpin may include an RNA loop. A hairpin can be formed by two complementary sequences within a single nucleic acid molecule that has folded or wrinkled together. In some embodiments, the hairpin includes a stem or a stem-loop structure.

[0041] As used herein, "region" refers to a conserved group of nucleic acids. A region may also be referred to as a "module" or "domain". Regions of gRNA may perform specific functions, for example, directing the endonuclease activity of the RNP as described in Briner AE et al., Molecular Cell 56: 333-339 (2014). Regions of gRNA are described in Tables 1-3.

[0042] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to, for example, a gRNA associated with a nuclease such as a Cas protein. In some embodiments, the RNP comprises Cas9 and gRNA.

[0043] As used herein, "stem-loop" refers to the secondary structure of nucleotides that forms a "stem" of base pairs ending in a loop of unpaired nucleic acid. The stem may be formed when the sequences of two regions of the same nucleic acid strand are at least partially complementary when read in opposite directions. As used herein, "loop" refers to a region of unpaired (i.e., non-complementary) nucleotides that may cap the stem. "Tetraloop" refers to a loop of four nucleotides. As used herein, the upper stem of the sgRNA may contain a tetraloop.

[0044] In certain embodiments involving dgRNA, the "stem" region as used herein refers to the secondary structure of nucleotides that forms a region of base pairs between a certain region of the crRNA and a certain region of the trRNA (e.g., the lower stem region and the upper stem region of each RNA). The "stem" region of the dgRNA may also be referred to in the art as the "flagpole" region.

[0045] As used herein, "treatment" encompasses any therapeutic administration or application for a disease in a subject, and also includes suppressing the disease, blocking its progression, alleviating one or more symptoms of the disease, curing the disease, or preventing recurrence of one or more symptoms of the disease.

[0046] 1. Types of Modifications A. 2'-O-Methyl Modification Modified sugars are thought to control the packing of the sugar rings of nucleotides, which are physical properties that affect oligonucleotide binding affinity, duplex formation, and interaction with nucleases for the complementary strand. Thus, substitutions on the sugar ring can alter the conformation and packing of these sugars. For example, 2'-O-methyl (2'-O-Me) modifications can increase the binding affinity and nuclease stability of oligonucleotides, but as shown in the examples, the effect of any modification at a given position in an oligonucleotide needs to be determined empirically.

[0047] The terms "mA", "mC", "mU", or "mG" may be used to represent nucleotides modified with 2'-O-Me.

[0048] Modification of ribonucleotides as 2'-O-methyl ribonucleotides can be depicted as follows:

Chemical formula

[0049] B. 2'-O-(2-methoxyethyl) modification In some embodiments, the modification may be 2'-O-(2-methoxyethyl) (2'-O-moe). Modification of ribonucleotides as 2'-O-moe ribonucleotides can be depicted as follows:

Chemical formula

[0050] The terms "moeA", "moeC", "moeU", or "moeG" may be used to represent nucleotides modified with 2'-O-moe.

[0051] C. 2'-fluoro modification Another chemical modification that has been shown to affect the sugar ring of nucleotides is halogen substitution. For example, 2'-fluoro (2'-F) substitution on the sugar ring of nucleotides can increase the binding affinity and nuclease stability of oligonucleotides.

[0052] In this application, the terms "fA", "fC", "fU", or "fG" may be used to represent nucleotides substituted with 2'-F.

[0053] The substitution of 2'-F can be depicted as follows:

Chemical formula

[0054] D. Phosphorothioate modification A phosphorothioate (PS) linkage or bond refers to a bond in which one non-bridging phosphoric acid oxygen is substituted by sulfur during a phosphodiester linkage, for example, during the bond between nucleotide bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may sometimes be referred to as S-oligos.

[0055] "*" may be used to depict PS modification. In this application, the terms A*, C*, U*, or G* may be used to represent nucleotides linked to the next (e.g., 3') nucleotide by a PS bond.

[0056] In this application, the terms "mA*", "mC*", "mU*", or "mG*" may be used to represent nucleotides substituted with 2'-O-Me and linked to the next (e.g., 3') nucleotide by a PS bond. Similarly, the terms "fA*", "fC*", "fU*", or "fG*" may be used to represent nucleotides substituted with 2'-F and linked to the next (e.g., 3') nucleotide by a PS bond. Equivalents of PS linkages or bonds are included in the embodiments described herein.

[0057] The following illustration shows the substitution of non-bridging phosphate oxygen by S- to generate a PS bond instead of a phosphodiester bond:

Chemical formula

[0058] E.G-C substitution In some embodiments, the gRNA is modified by sequence substitution without chemical modification. In some embodiments, the modified gRNA is engineered using G-C pairing (e.g., in the lower stem region and / or the upper stem region) that is not found in the parental gRNA sequence. In some embodiments, the modified gRNA is engineered using G-U mismatches ( "GU wobble" or mismatched pairing) that are not found in the parental gRNA sequence.

[0059] F. Inverted abasic modification An abasic nucleotide refers to a nucleotide lacking a nitrogenous base. The following figure depicts an oligonucleotide having an abasic (also known as apurinic) site lacking a base:

Chemical formula

[0060] An inverted base refers to a base having a linkage reversed from the normal 5' to 3' linkage (i.e., either a 5' to 5' linkage or a 3' to 3' linkage). For example:

Chemical formula

[0061] Apurinic nucleotides can be ligated by inverse ligation. For example, an apurinic nucleotide may be ligated to the 5' nucleotide at the terminus via a 5' to 5' ligation, or an apurinic nucleotide may be ligated to the 3' nucleotide at the terminus via a 3' to 3' ligation. Apurinic nucleotides in either the 5' or 3' nucleotide at the terminus may also be referred to as an inverted apurinic end cap. In the present application, the term "invd" refers to an inverted apurinic nucleotide ligation.

[0062] The above modifications and their equivalents are included within the scope of the embodiments described herein.

[0063] 2. Guide RNA Composition Compositions containing guide RNA are included. In some embodiments, the guide RNA includes a trRNA. In some embodiments, the guide RNA includes a crRNA. In some embodiments, the guide RNA includes a crRNA and a trRNA. In some embodiments, the guide RNA includes a crRNA and a trRNA on one RNA molecule as a sgRNA. In some embodiments, the guide RNA includes a crRNA and a trRNA on two RNA molecules as a dgRNA. In a dgRNA, the two RNA molecules may associate via base pairing.

[0064] 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 the "spacer" region described in Briner AE et al., Molecular Cell 56: 333-339 (2014) for sgRNA (although 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 the spacer region, may be associated with crRNA, trRNA, sgRNA, and / or dgRNA. The spacer region is sometimes also referred to herein and elsewhere as the "guide region", "guide domain" or "targeting domain". As used herein, "target sequence" refers to the sequence of a nucleic acid to which the guide region / domain directs a nuclease for cleavage. In some embodiments, the spyCas9 protein may be directed by the guide region / domain to the target sequence of the target nucleic acid by nucleotides present in the spacer region. In some embodiments, the guide RNA does not comprise a spacer region.

[0065] In some embodiments, the guide RNAs described herein comprise or consist of any of the sequences shown in Table 4. However, it should be noted that when the sequence represents the guide / spacer region, the composition may or may not include this region. Further, guide RNAs are included that contain any modification of any of the sequences shown in Table 4 and identified by SEQ ID NOs in Table 4. That is, the nucleotides may be the same or different, but the modification pattern shown may be the same as or similar to the modification pattern of the guide sequences in Table 4. The modification pattern includes the relative position and identity of the modification of the gRNA or the region of the gRNA (e.g., 5' terminal region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3' terminal region). In some embodiments, the modification pattern contains at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the modification of any one of the sequences shown in the sequence column of Table 4 or the modification over 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 one 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 over one or more regions of the sequences shown in Table 4, such as the 5' terminal region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, and / or 3' terminal region. For example, in some embodiments, guide RNAs are included in which 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 over the 5' terminal region.In some embodiments, guide RNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across the lower stem. In some embodiments, guide RNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across the bulge. In some embodiments, guide RNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across the upper stem. In some embodiments, guide RNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across the nexus. In some embodiments, guide RNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across hairpin 1. In some embodiments, guide RNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across hairpin 2. In some embodiments, guide RNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across the 3' terminus. In some embodiments, the modification pattern is different from the modification pattern of the sequence of Table 4, or a region of such a sequence (e.g., 5' terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3' terminus) at 0, 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, the gRNA includes modifications that are different from the modifications of the sequence of Table 4 at 0, 1, 2, 3, 4, 5, or 6 nucleotides.In some embodiments, the gRNA comprises modifications different from those of the regions of the sequences in Table 4 at 0, 1, 2, 3, 4, 5, or 6 nucleotides (e.g., 5' end, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3' end).

[0066] 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) linkages between nucleotides.

[0067] In some embodiments, the gRNA comprises a 5' end modification, a 3' end modification, or both a 5' end modification and a 3' end modification. In some embodiments, the 5' end modification comprises phosphorothioate (PS) linkages between nucleotides. In some embodiments, the 5' end modification comprises 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), and / or 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the 5' end modification comprises at least one phosphorothioate (PS) linkage and one or more of 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), and / or 2'-fluoro (2'-F) modified nucleotides. The end modification may comprise phosphorothioate (PS), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), and / or 2'-fluoro (2'-F) modifications. Equivalent end modifications are also encompassed by the embodiments described herein. In some embodiments, the gRNA comprises an end modification in combination with a modification of one or more regions of the gRNA.

[0068] A. Compositions of sgRNA In some embodiments, the compositions and methods of the invention include a gRNA comprising a crRNA and a trRNA that direct a nuclease, such as Cas9, to a target DNA sequence. In some embodiments, the gRNAs described herein may be associated on a single RNA molecule (single guide RNA or sgRNA).

[0069] In some embodiments, the invention includes an sgRNA comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 228 - 332.

[0070] In some embodiments, an sgRNA is provided that comprises any one of the modified sequences of SEQ ID NOs: 235 - 240, 265 - 285, and 309 - 329. In some embodiments, an sgRNA that comprises any one of the modified sequences of SEQ ID NOs: 235 - 240, 265 - 285, and 309 - 329 and that is complementary to a target sequence and further comprises a 5’ “spacer” sequence (“guide sequence”) that directs Cas9 to the target for its cleavage is included. In some instances, the invention includes 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 the nucleic acids of SEQ ID NOs: 235 - 240, 265 - 285, and 309 - 329 and having a modification pattern that is identical to the modification pattern shown in the reference sequence identifier, and includes an sgRNA.

[0071] 1. Domains of sgRNA Briner AE et al., Molecular Cell 56: 333 - 339 (2014) describes the functional domains of an sgRNA (referred to herein as “domains”), such as a “spacer” domain involved in targeting, a “lower stem,” a “bulge,” an “upper stem” (which may include a tetraloop), a “nexus,” and a “hairpin 1” domain and a “hairpin 2” domain. See Briner et al., page 334, FIG. 1A.

[0072] Table 1 and FIG. 21A provide an illustration of the domains of the sgRNA used herein. In Table 1, "n" between regions represents a variable number of nucleotides, for example, 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, n is equal to 0. In some embodiments, n is equal to 1. [Table 2]

[0073] a) 5’ End Terminal region In some embodiments, the sgRNA contains nucleotides at the 5' end as shown in Table 1. In some embodiments, the 5' end of the sgRNA contains a spacer or guide region that functions to direct the Cas protein to the target nucleotide sequence. In some embodiments, the 5' end does not contain a spacer or guide region. In some embodiments, the 5' end contains a spacer and additional nucleotides that do not function to direct the Cas protein to the target nucleotide region.

[0074] In some embodiments, the guide region contains 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 contains 20 nucleotides. In some embodiments, the guide region may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides. In some embodiments, the guide region may contain 17 nucleotides. In some embodiments, the guide region may contain 18 nucleotides. In some embodiments, the guide region may contain 19 nucleotides.

[0075] In some embodiments, the selection of the guide region is determined based on a target sequence within the gene of interest for editing. For example, in some embodiments, the sgRNA comprises a guide region complementary to the target sequence of the gene of interest.

[0076] 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 of the gene of interest may be 100% complementary or identical. In other embodiments, the guide region of the sgRNA and the target region 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 when the full length of the target sequence is at least about 17, 18, 19, 20, or more base pairs. In some embodiments, the guide region of the sgRNA and the target region of the gene of interest may contain 1 - 6 mismatches when the guide sequence contains at least about 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide region of the sgRNA and the target region of the gene of interest may contain 1, 2, 3, 4, 5, or 6 mismatches when the guide sequence contains about 20 nucleotides. The 5' end may contain nucleotides that are not considered part of the guide region (i.e., do not function to direct the Cas9 protein to the target nucleic acid).

[0077] b) Lower stem In some embodiments, the sgRNA comprises a lower stem (LS) region separated by a bulge and an upper stem region when viewed linearly. See Table 1.

[0078] In some embodiments, the lower stem region comprises from 1 to 12 nucleotides. For example, in one embodiment, the lower stem region comprises LS1-LS12. In some embodiments, the lower stem region comprises fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the lower stem region comprises more nucleotides than shown in Table 1 and FIG. 21A. When the lower stem region comprises fewer or more nucleotides than shown in the schematics of Table 1 and FIG. 21A, the modification pattern should be maintained, as will be apparent to those skilled in the art.

[0079] In some embodiments, the lower stem region has nucleotides whose nucleic acid sequences are complementary when read in the opposite direction. In some embodiments, the complementarity in the nucleic acid sequence of the lower stem leads to the secondary structure of the stem in the sgRNA (e.g., the regions may base pair with each other). In some embodiments, the lower stem region may not be completely complementary to each other when read in the opposite direction.

[0080] c) Bulge In some embodiments, the sgRNA comprises a bulge region containing 6 nucleotides B1-B6. When viewed linearly, the bulge region is separated into two regions. See Table 1. In some embodiments, the bulge region contains 6 nucleotides, with the first 2 nucleotides followed by the upper stem region, and the last 4 nucleotides of the bulge followed by the upper stem region. In some embodiments, the bulge region comprises fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the bulge region comprises more nucleotides than shown in Table 1 and FIG. 21A. When the bulge region comprises fewer or more nucleotides than shown in the schematics of Table 1 and FIG. 21A, the modification pattern should be maintained, as will be apparent to those skilled in the art.

[0081] In some embodiments, the presence of the bulge results in a directional kink between the upper stem module and the lower stem module in the sgRNA.

[0082] d) Upper stem In some embodiments, the sgRNA includes an upper stem region that includes 12 nucleotides. In some embodiments, the upper stem region includes a loop sequence. In some cases, the loop is a tetraloop (a loop consisting of 4 nucleotides).

[0083] In some embodiments, the upper stem region includes fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the upper stem region includes more nucleotides than shown in Table 1 and FIG. 21A. When the upper stem region includes fewer or more nucleotides than shown in the schematics of Table 1 and FIG. 21A, the modification pattern should be maintained, as will be apparent to those skilled in the art.

[0084] In some embodiments, the upper stem region has nucleotides whose nucleic acid sequences are complementary when read in the opposite direction. In some embodiments, the complementarity in the nucleic acid sequence of the upper stem leads to the secondary structure of the stem in the sgRNA (e.g., the regions may base pair with each other). In some embodiments, the upper stem region may not be completely complementary to each other when read in the opposite direction.

[0085] e) Nexus In some embodiments, the sgRNA includes a nexus region located between the lower stem region and the hairpin 1 region. In some embodiments, the nexus includes 18 nucleotides. In some embodiments, the nexus region includes nucleotides N1 to N18, as shown in Table 1 and FIG. 21A.

[0086] In some embodiments, the nexus region includes fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the nexus region includes more nucleotides than shown in Table 1 and FIG. 21A. When the nexus region includes fewer or more nucleotides than shown in the schematics of Table 1 and FIG. 21A, the modification pattern should be maintained, as will be apparent to those skilled in the art.

[0087] In some embodiments, the nexus region has nucleotides whose nucleic acid sequences are complementary when read in opposite directions. In some embodiments, the complementarity in the nucleic acid sequence leads to the secondary structure of the stem and / or stem-loop in the sgRNA (e.g., certain nucleotides in the nexus region may base pair with each other). In some embodiments, the nexus region may not be completely complementary to each other when read in opposite directions.

[0088] f) Hairpin In some embodiments, the sgRNA includes one or more hairpin regions. In some embodiments, the hairpin region is downstream (e.g., 3’) of the nexus region. In some embodiments, the region of nucleotides immediately downstream of the nexus region is referred to as “hairpin 1” or “H1”. In some embodiments, the region of nucleotides 3’ of hairpin 1 is referred to as “hairpin 2” or “H2”. In some embodiments, the hairpin region includes hairpin 1 and hairpin 2. In some embodiments, the sgRNA includes only hairpin 1 or hairpin 2.

[0089] In some embodiments, the hairpin 1 region includes 12 nucleic acids immediately downstream of the nexus region. In some embodiments, the hairpin 1 region includes nucleotides H1-1 to H1-12 as shown in Table 1 and FIG. 21A.

[0090] In some embodiments, the hairpin 2 region includes 15 nucleic acids downstream of the hairpin 1 region. In some embodiments, the hairpin 2 region includes nucleotides H2-1 to H2-15 as shown in Table 1 and FIG. 21A.

[0091] In some embodiments, one or more nucleotides are present between the hairpin 1 region and the hairpin 2 region. The one or more nucleotides between the hairpin 1 region and the hairpin 2 region may be modified or unmodified. In some embodiments, hairpin 1 and hairpin 2 are separated by one nucleotide. In some embodiments, the hairpin region contains fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the hairpin region contains more nucleotides than shown in Table 1 and FIG. 21A. When the hairpin region contains fewer or more nucleotides than shown in the schematics of Table 1 and FIG. 21A, the modification pattern should be maintained, as will be apparent to those skilled in the art.

[0092] In some embodiments, the hairpin region has nucleotides whose nucleic acid sequences are complementary when read in the opposite direction. In some embodiments, the hairpin regions may not be completely complementary to each other when read in the opposite direction (e.g., the top or loop of the hairpin contains unpaired nucleotides).

[0093] In some embodiments, the sgRNA contains a substitution of hairpin 1 at nucleotide "n", where "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 replaced by two nucleotides.

[0094] g) 3’ End Terminal region In some embodiments, the sgRNA includes nucleotides after the hairpin region(s). In some embodiments, the 3' terminal region includes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments, the 3' terminal region includes 1, 2, 3, or 4 nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments, the 3' terminal region includes 4 nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments, the 3' terminal region includes 1, 2, or 3 nucleotides that do not associate with the secondary structure of the hairpin.

[0095] 2. Modification of sgRNA In some embodiments, the invention includes an sgRNA comprising one or more modifications within one or more of the following regions: nucleotides at the 5' end; the lower stem region; the bulge region; the upper stem region; the nexus region; the hairpin 1 region; the hairpin 2 region; and nucleotides at the 3' end.

[0096] In some embodiments, the modification includes 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the modification includes 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides. In some embodiments, the modification includes 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the modification includes phosphorothioate (PS) linkages between nucleotides.

[0097] In some embodiments, the sgRNA comprises a modification at one, two, three, or four of the first four nucleotides at its 5' end. In some embodiments, the first three or four nucleotides at the 5' terminus, and the last three or four nucleotides at the 3' terminus are modified. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by phosphorothioate (PS) bonds. 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.

[0098] In some embodiments, the sgRNA comprises a modification at one, two, three, or four of the first four nucleotides at its 5' end. In some embodiments, the sgRNA comprises a modification at one, two, three, or four of the first four nucleotides at its 3' end. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise 2'-O-Me or 2'-O-moe modifications.

[0099] In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise 2'-F modifications.

[0100] In some embodiments, sgRNAs are provided where LS1, LS6, LS7, LS8, LS11, and LS12 are modified with 2'-O-Me. In some embodiments, each of the nucleotides in the bulge 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 nexus 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.

[0101] In some embodiments, the sgRNA comprises 2'-O-Me modified nucleotides at the following nucleotides: the first 3 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 nexus region; each of the nucleotides in the hairpin 1 region; each of the nucleotides in the hairpin 2 region; and the last 4 nucleotides at the 3' end.

[0102] In some embodiments, the sgRNA further comprises three phosphorothioate (PS) linkages that link the first four nucleotides at the 5' end and three PS linkages that link 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, third, and fourth nucleotides from the end at the 3' end are modified with 2'-F.

[0103] In some embodiments, provided is a single guide RNA (sgRNA) comprising a 2'-F modified nucleic acid in the following nucleotides: LS9 and LS10 in the lower stem region; N15, N16, N17, and N18 in the nexus 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 2'-F modified nucleotides at the second, third, and fourth nucleotides from the end at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) linkages that link the first four nucleotides at the 5' end and three PS linkages that link 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 in three of the last four nucleotides at the 3' end.

[0104] In some embodiments, a single guide RNA (sgRNA) is provided that includes 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, 2'-O-Me modified nucleotides at LS1 and LS6, 2'-O-Me modified nucleotides at US1-US12, 2'-O-Me modified nucleotides at H1-1 - H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides at H2-1 - H2-15, and 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end. In some embodiments, the sgRNA further includes 3 phosphorothioate (PS) linkages that link the first 4 nucleotides at the 5' end and 3 PS linkages that link the last 4 nucleotides at the 3' end.

[0105] In some embodiments, a single guide RNA (sgRNA) is provided that includes 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, 2'-F modified nucleotides at LS1-LS6, 2'-O-Me modified nucleotides at US1-US12, 2'-O-Me modified nucleotides at H1-1 - H1-12, 2'-O-Me modified nucleotides at "n" between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides at H2-1 - H2-15, and 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end. In some embodiments, the sgRNA further includes 3 phosphorothioate (PS) linkages that link the first 4 nucleotides at the 5' end and 3 PS linkages that link the last 4 nucleotides at the 3' end.

[0106] In some embodiments, a single-guide RNA (sgRNA) is provided, which includes 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, 2'-F modified nucleotides at LS2-LS5, 2'-O-Me modified nucleotides at LS1 and LS6, 2'-O-Me modified nucleotides at US1-US12, 2'-O-Me modified nucleotides at H1-1 - H1-12, 2'-O-Me modified nucleotides at "n" between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides at H2-1 - H2-15, and 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end. In some embodiments, the sgRNA further includes 3 phosphorothioate (PS) bonds that link the first 4 nucleotides at the 5' end and 3 PS bonds that link the last 4 nucleotides at the 3' end.

[0107] In some embodiments, a single-guide RNA (sgRNA) is provided, which includes 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, 2'-O-Me modified nucleotides at US1-US12, 2'-O-Me modified nucleotides at LS7, LS8, LS11, and LS12, 2'-O-Me modified nucleotides at H1-1 - H1-12, 2'-O-Me modified nucleotides at "n" between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides at H2-1 - H2-15, and 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end. In some embodiments, the sgRNA further includes 3 phosphorothioate (PS) bonds that link the first 4 nucleotides at the 5' end and 3 PS bonds that link the last 4 nucleotides at the 3' end.

[0108] In some embodiments, a single-guide RNA (sgRNA) is provided that includes 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in LS8, LS10, and LS12, 2'-O-F modified nucleotides in LS7, LS9, and LS11, 2'-O-Me modified nucleotides in H1-1 - H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1 - H2-15, and 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end. In some embodiments, the sgRNA further includes 3 phosphorothioate (PS) linkages that link the first 4 nucleotides at the 5' end and 3 PS linkages that link the last 4 nucleotides at the 3' end.

[0109] In some embodiments, a single-guide RNA (sgRNA) is provided that includes 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end, 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in H1-1 - H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1 - H2-15, and 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' end. In some embodiments, the sgRNA further includes 3 phosphorothioate (PS) linkages that link the first 4 nucleotides at the 5' end and 3 PS linkages that link the last 4 nucleotides at the 3' end.

[0110] In some embodiments, a single-guide RNA (sgRNA) is provided that 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-US12, 2'-O-Me modified nucleotides at H1-1 - H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides at H2-1 - 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) linkages that link the first four nucleotides at the 5' end and three PS linkages that link the last four nucleotides at the 3' end.

[0111] In some embodiments, a single-guide RNA (sgRNA) is provided that includes 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides at US1-US12, 2'-O-Me modified nucleotides at H1-1 - H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides at H2-1 - H2-8, 2'-F modified nucleotides at H2-9 - H2-15, 2'-F modified nucleotides at the second last, third last, and fourth 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 includes three phosphorothioate (PS) linkages that link the first four nucleotides at the 5' end and three PS linkages that link the last four nucleotides at the 3' end.

[0112] In some embodiments, a single-guide RNA (sgRNA) is provided that includes 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12, 2'-F modified nucleotides in H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11, 2'-F modified nucleotides between hairpin 1 and hairpin 2, 2'-F modified nucleotides in H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14, 2'-O-Me modified nucleotides in H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15, 2'-F modified nucleotides at the second last and fourth last nucleotides at the 3' end, and 2'-O-Me modified nucleotides at the third last and last nucleotides at the 3' end. In some embodiments, the sgRNA further includes three phosphorothioate (PS) linkages that link the first four nucleotides at the 5' end and three PS linkages that link the last four nucleotides at the 3' end.

[0113] In some embodiments, a single-guide RNA (sgRNA) is disclosed herein that contains 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 contains 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 that link the first four nucleotides at the 5' terminus and three PS bonds that link the last four nucleotides at the 3' terminus. In some embodiments, the sgRNA further comprises 2'-O-Me modified nucleotides at the last and the third-to-last nucleotides at the 3' terminus, and further comprises 2'-F modified nucleotides at the second-to-last and the third-to-last nucleotides at the 3' terminus.

[0114] In some embodiments, an sgRNA comprising any one of the nucleic acids of SEQ ID NOs: 228 - 232 is disclosed herein. In some embodiments, an sgRNA comprising any one of the nucleic acids of SEQ ID NOs: 235 - 240, 265 - 285, and 309 - 329 is disclosed herein. In some embodiments, an sgRNA is disclosed herein 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 any one of the nucleic acids of SEQ ID NOs: 235 - 240, 265 - 285, and 309 - 329, and the modification pattern is identical to the modification pattern shown in the reference sequence identifier. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds that link the first four nucleotides at the 5' terminus and three PS bonds that link the last four nucleotides at the 3' terminus.

[0115] In some embodiments, an sgRNA is provided that includes a 5'-end modification and one or more modifications in one or more of an upper stem region; a hairpin 1 region; and a hairpin 2 region, wherein the 5'-end modification includes at least two phosphorothioate linkages within the first 7 nucleotides at the 5'-terminus.

[0116] In some embodiments, an sgRNA is provided that includes a 5'-end modification and one or more modifications in one or more of an upper stem region; a hairpin 1 region; and a hairpin 2 region, wherein the 5'-end modification includes one or more phosphorothioate linkages at the 5'-terminus of the RNA. In some embodiments, one or more phosphorothioate bonds link the nucleotides at the 5'-terminus.

[0117] In some embodiments, an sgRNA is provided that includes a 5'-end modification and one or more modifications in one or more of an upper stem region; a hairpin 1 region; and a hairpin 2 region, wherein the 5'-end modification includes one or more phosphorothioate linkages within the first 7 nucleotides at the 5'-terminus.

[0118] In some embodiments, an sgRNA is provided that includes any one of the modified sgRNA sequences of SEQ ID NOs: 228 to 332.

[0119] In some embodiments, an sgRNA is provided that includes or consists of any one of the modified sgRNA sequences of SEQ ID NOs: 235 to 240, 265 to 285, and 309 to 329.

[0120] In some embodiments, the present invention includes an sgRNA that includes any one of the modified sequences of SEQ ID NOs: 235 to 240, 265 to 285, and 309 to 329, wherein the sgRNA is at least partially complementary to a target sequence and further includes a 5'-spacer sequence that directs Cas9 to cleave at that target.

[0121] In some embodiments, the present invention includes an sgRNA comprising a nucleotide having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one nucleotide of SEQ ID NOs: 235-240, 265-285, and 309-329, and having a modification pattern identical to the modification pattern shown in the reference sequence identifier. That is, the nucleotides A, U, C, and G may be 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% different compared to those shown in the sequence, but the modification remains unchanged.

[0122] In some embodiments, the present invention includes an sgRNA comprising one or more modifications within one or more of the following regions: nucleotides at the 5' end; lower stem region; bulge region; upper stem region; nexus region; hairpin 1 region; hairpin 2 region; and nucleotides at the 3' end.

[0123] In some embodiments, the modification includes 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the modification includes 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the modification includes phosphorothioate (PS) bonds between nucleotides. In some embodiments, the modification includes inverted abasic nucleotides.

[0124] In some embodiments, the sgRNA is provided with 2'-O-Me modified nucleotides at the first 3 nucleotides in the 5' terminus, LS1, LS6, LS7, LS8, LS11, and LS12 in the lower stem, B1 and B2 in the bulge region, each of the nucleotides in the upper stem region, N16, N17, and N18 in the nexus region, each of the nucleotides in the hairpin 1 region, 1 nucleotide between hairpin 1 and hairpin 2, each of the nucleotides in the hairpin 2 region, and the last 4 nucleotides at the 3' terminus. In one embodiment, the sgRNA further comprises 3 PS bonds between the first 4 nucleotides at the 5' terminus and 3 PS bonds between the last 4 nucleotides at the 3' terminus.

[0125] In some embodiments, the sgRNA is provided with 2'-O-Me modified nucleotides at the first 3 nucleotides in the 5' terminus, LS1, LS6, LS7, LS8, LS11, and LS12 in the lower stem, B1 - B6 in the bulge region, each of the nucleotides in the upper stem region, N16, N17, and N18 in the nexus region, each of the nucleotides in the hairpin 1 region, 1 nucleotide between hairpin 1 and hairpin 2, each of the nucleotides in the hairpin 2 region, and the last 4 nucleotides at the 3' terminus. In one embodiment, the sgRNA further comprises 3 PS bonds between the first 4 nucleotides at the 5' terminus and 3 PS bonds between the last 4 nucleotides at the 3' terminus.

[0126] In some embodiments, the sgRNA is provided with 2'-F modified nucleotides at LS9 and LS10 in the lower stem, 15 - N18 in the nexus region, H2 - 9 - HS - 15 in the hairpin 2 region, and the second last, third last, and fourth last nucleotides in the 3' terminal region.

[0127] In some embodiments, the sgRNA is provided with 2'-F modified nucleotides in each nucleotide in the lower stem, 15 to N18 in the nexus region, H2-9 to HS-15 in the hairpin 2 region, and the second last, third last, and fourth last nucleotides in the 3' terminal region.

[0128] In some embodiments, the single guide RNA (sgRNA) is provided with 2'-O-Me modified nucleotides in 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 last nucleotides at the 3' terminus, and with 2'-F modification in 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 last and fourth last nucleotides at the 3' terminus.

[0129] Each of the following embodiments is included: Embodiment 01. A single guide RNA (sgRNA) comprising one or more modifications in one or more of the following regions: a. The 5' terminus and b. The lower stem region and c. The bulge region and d. The upper stem region and e. The nexus region and f. The hairpin 1 region and g. The hairpin 2 region and h. The 3' terminus, the sgRNA. Embodiment 02. The sgRNA of Embodiment 1, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. Embodiment 03. The sgRNA of Embodiment 1, wherein the modification comprises a 2'-fluoro (2'-F) modified nucleotide. Embodiment 04. The sgRNA of Embodiment 1, wherein the modification comprises phosphorothioate (PS) bonds between nucleotides. Embodiment 05. The sgRNA of any one of Embodiments 1 to 3, wherein the first 3 or 4 nucleotides at the 5'-terminus and the last 3 or 4 nucleotides at the 3'-terminus are modified. Embodiment 06. The sgRNA of any one of Embodiments 1 to 5, wherein the first 4 nucleotides at the 5'-terminus and the last 4 nucleotides at the 3'-terminus are linked by phosphorothioate (PS) bonds. Embodiment 07. The sgRNA of Embodiment 5, wherein the modification comprises 2'-O-Me. Embodiment 08. The sgRNA of Embodiment 5, wherein the modification comprises 2'-F. Embodiment 09. The sgRNA of any one of Embodiments 1 to 7, wherein the first 4 nucleotides at the 5'-terminus and the last 4 nucleotides at the 3'-terminus are linked by PS bonds, and the first 3 nucleotides at the 5'-terminus and the last 3 nucleotides at the 3'-terminus comprise 2'-O-Me modification. Embodiment 10. The sgRNA of any one of Embodiments 1 to 8, wherein the first 4 nucleotides at the 5'-terminus and the last 4 nucleotides at the 3'-terminus are linked by PS bonds, and the first 3 nucleotides at the 5'-terminus and the last 3 nucleotides at the 3'-terminus comprise 2'-F modification. 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. Embodiment 12. The sgRNA of any one of Embodiments 1 to 11, wherein each of the nucleotides in the bulge region is modified with 2'-O-Me. Embodiment 13. An sgRNA of any one of Embodiments 1 to 12, wherein each nucleotide in the upper stem region is modified with 2'-O-Me. Embodiment 14. An sgRNA of any one of Embodiments 1 to 13, wherein N16, N17, and N18 in the nexus region are modified with 2'-O-Me. Embodiment 15. An sgRNA of any one of Embodiments 1 to 14, wherein each nucleotide in the hairpin 1 region is modified with 2'-O-Me. Embodiment 16. An sgRNA of any one of Embodiments 1 to 15, wherein each nucleotide in the hairpin 2 region is modified with 2'-O-Me. Embodiment 17. A single guide RNA (sgRNA) comprising the following nucleotides: a. The first 3 nucleotides at the 5' end, and b. The LS1, LS6, LS7, LS8, LS11, and LS12 regions in the lower stem region, and c. B1 and B2 in the bulge region, and d. Each nucleotide in the upper stem region, and e. N16, N17, and N18 in the nexus region, and f. Each nucleotide in the hairpin 1 region, and g. Each nucleotide in the hairpin 2 region, and h. The last 4 nucleotides at the 3' end, and wherein the sgRNA comprises a 2'-O-Me modified nucleic acid. Embodiment 18. The sgRNA of Embodiment 17, wherein B3 to B6 are modified with 2'-O-Me. Embodiment 19. The sgRNA of Embodiment 17, further comprising 3 phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end and 3 PS bonds connecting the last 4 nucleotides at the 3' end. Embodiment 20. An sgRNA of any one of Embodiments 1-10, wherein LS9 and LS10 are modified with 2'-F. Embodiment 21. An sgRNA of any one of Embodiments 1-10 and 20, wherein N15, N16, N17, and N18 are modified with 2'-F. Embodiment 22. An sgRNA of any one of Embodiments 1-10 and 20-21, wherein H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 are modified with 2'-F. Embodiment 23. An sgRNA of any one of Embodiments 1-10 and 21-22, wherein the second last, third last, and fourth last nucleotides at the 3' terminus are modified with 2'-F. Embodiment 24. A single guide RNA (sgRNA) at the following positions: a. LS9 and LS10 in the lower stem region, b. N15, N16, N17, and N18 in the nexus region, c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 in the hairpin 2 region, containing 2'-F modified nucleotides, a single guide RNA (sgRNA). Embodiment 25. The sgRNA of Embodiment 24, further containing 2'-F modified nucleotides at the second last, third last, and fourth last nucleotides at the 3' terminus. Embodiment 26. The sgRNA of any one of Embodiments 24 or 25, further containing three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' terminus. Embodiment 27. An sgRNA according to any one of Embodiments 24 to 26, further comprising a 2'-O-Me or 2'-F modified nucleotide in the first 3 nucleotides at the 5' end and further comprising a 2'-O-Me or 2'-F modified nucleotide in 3 of the last 4 nucleotides at the 3' end. Embodiment 28. A single guide RNA (sgRNA), a. A 2'-O-Me modified nucleotide in the first 3 nucleotides at the 5' end, b. 2'-O-Me modified nucleotides in LS1 and LS6, c. 2'-O-Me modified nucleotides in US1 to US12, d. 2'-O-Me modified nucleotides in H1-1 to H1-12, e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, f. 2'-O-Me modified nucleotides in H2-1 to H2-15, g. A 2'-O-Me modified nucleotide in the last 4 nucleotides at the 3' end, and comprising the sgRNA. Embodiment 29. The sgRNA of Embodiment 28, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end and three PS bonds connecting the last 4 nucleotides at the 3' end. Embodiment 30. A single guide RNA (sgRNA), a. A 2'-O-Me modified nucleotide in the first 3 nucleotides at the 5' end, b. 2'-F modified nucleotides in LS1 to LS6, c. 2'-O-Me modified nucleotides in US1 to US12, d. 2'-O-Me modified nucleotides in H1-1 to H1-12, e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, f. 2'-O-Me modified nucleotides at H2-1 to H2-15, and g. 2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, an sgRNA comprising. Embodiment 31. The sgRNA of Embodiment 30, further comprising three phosphorothioate (PS) bonds linking the first 4 nucleotides at the 5' terminus and three PS bonds linking the last 4 nucleotides at the 3' terminus. Embodiment 32. A single guide RNA (sgRNA) comprising: a. 2'-O-Me modified nucleotides in the first 3 nucleotides at the 5' terminus, and b. 2'-F modified nucleotides at LS2 to LS5, and c. 2'-O-Me modified nucleotides at LS1 and LS6, and d. 2'-O-Me modified nucleotides at US1 to US12, and e. 2'-O-Me modified nucleotides at H1-1 to H1-12, and f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and g. 2'-O-Me modified nucleotides at H2-1 to H2-15, and h. 2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, a single guide RNA (sgRNA) comprising. Embodiment 33. The sgRNA of Embodiment 32, further comprising three phosphorothioate (PS) bonds linking the first 4 nucleotides at the 5' terminus and three PS bonds linking the last 4 nucleotides at the 3' terminus. Embodiment 34. A single guide RNA (sgRNA) comprising: a. 2'-O-Me modified nucleotides in the first 3 nucleotides at the 5' terminus, and b. 2'-O-Me modified nucleotides at US1 to US12, and c. 2'-O-Me modified nucleotides in LS7, LS8, LS11, and LS12, and d. 2'-O-Me modified nucleotides in H1-1 to H1-12, and e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and f. 2'-O-Me modified nucleotides in H2-1 to H2-15, and g. 2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, an sgRNA comprising the same. Embodiment 35. The sgRNA of Embodiment 34, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' terminus. Embodiment 36. A single guide RNA (sgRNA), a. 2'-O-Me modified nucleotides in the first 3 nucleotides at the 5' terminus, and b. 2'-O-Me modified nucleotides in US1 to US12, and c. 2'-O-Me modified nucleotides in LS7, LS8, LS11, and LS12, and d. 2'-F modified nucleotides in LS9 and LS10, and e. 2'-O-Me modified nucleotides in H1-1 to H1-12, and f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, and g. 2'-O-Me modified nucleotides in H2-1 to H2-15, and h. 2'-O-Me modified nucleotides in the last 4 nucleotides at the 3' terminus, an sgRNA comprising the same. Embodiment 37. The sgRNA of Embodiment 36, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' terminus. Embodiment 38. A single guide RNA (sgRNA) comprising: a. 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end; b. 2'-O-Me modified nucleotides in US1-US12; c. 2'-O-Me modified nucleotides in LS8, LS10, and LS12; d. 2'-O-F modified nucleotides in LS7, LS9, and LS11; e. 2'-O-Me modified nucleotides in H1-1~H1-12; f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; g. 2'-O-Me modified nucleotides in H2-1~H2-15; h. 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. 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. Embodiment 40. A single guide RNA (sgRNA) comprising: a. 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end; b. 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12; c. 2'-O-Me modified nucleotides in US1-US12; d. 2'-O-Me modified nucleotides in H1-1~H1-12; e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; f. 2'-O-Me modified nucleotides in H2-1~H2-15; g. 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. Embodiment 41. The sgRNA of Embodiment 40, further comprising three phosphorothioate (PS) bonds linking the first four nucleotides at the 5' end and three PS bonds linking the last four nucleotides at the 3' end. Embodiment 42. A single guide RNA (sgRNA), comprising: a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; b. 2'-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and LS12; c. 2'-F modified nucleotides at LS9 and LS10; d. 2'-O-Me modified nucleotides at US1-US12; e. 2'-O-Me modified nucleotides at H1-1-H1-12; f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; g. 2'-O-Me modified nucleotides at H2-1-H2-15; h. 2'-O-Me modified nucleotides at the last four nucleotides at the 3' end. Embodiment 43. The sgRNA of Embodiment 43, further comprising three phosphorothioate (PS) bonds linking the first four nucleotides at the 5' end and three PS bonds linking the last four nucleotides at the 3' end. Embodiment 44. A single guide RNA (sgRNA), comprising: a. 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end; b. 2'-O-Me modified nucleotides at US1-US12; c. 2'-O-Me modified nucleotides at H1-1-H1-12; d. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; e. The 2'-O-Me modified nucleotides in H2-1 to H2-8, and f. The 2'-F modified nucleotides in H2-9 to H2-15, and g. The 2'-F modified nucleotides in the second last, third last, and fourth last nucleotides at the 3' terminus, and h. The 2'-O-Me modified nucleotide in the last nucleotide at the 3' terminus, an sgRNA comprising the same. Embodiment 45. The sgRNA of Embodiment 44, further comprising 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. Embodiment 46. A single guide RNA (sgRNA), a. The 2'-O-Me modified nucleotides in the first three nucleotides at the 5' terminus, and b. The 2'-O-Me modified nucleotides in US1 to US12, and c. The 2'-O-Me modified nucleotides in H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12, and d. The 2'-F modified nucleotides in H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11, and e. The 2'-F modified nucleotides between hairpin 1 and hairpin 2, and f. The 2'-F modified nucleotides in H2-2, H2-4, H2-6, H2-8, H2-10, H2-12; and H2-14, and g. The 2'-O-Me modified nucleotides in H2-1, H2-3, H2-5, H2-7, H2-9, H2-11; H2-13, and H2-15, and h. The 2'-F modified nucleotides in the second last and fourth last nucleotides at the 3' terminus, and i. The 2'-O-Me modified nucleotides in the third last and last nucleotides at the 3' terminus, an sgRNA comprising the same. Embodiment 47. The sgRNA of embodiment 46, further comprising three phosphorothioate (PS) linkages that link the first four nucleotides at the 5'-terminus and three PS linkages that link the last four nucleotides at the 3'-terminus. Embodiment 48. A single guide RNA (sgRNA), a. 2'-O-Me modified 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 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, the sgRNA comprising the same. Embodiment 49. The sgRNA of embodiment 48, further comprising three phosphorothioate (PS) linkages that link the first four nucleotides at the 5'-terminus and three PS linkages that link the last four nucleotides at the 3'-terminus. Embodiment 50. a. 2'-O-Me modified nucleotides at the last and the third last nucleotides at the 3'-terminus, and b. 2'-F modified nucleotides at the second last and the third last nucleotides at the 3'-terminus, the sgRNA of any one of embodiments 48 to 49 further comprising the same. Embodiment 51. The sgRNA comprising any one nucleic acid of SEQ ID NOs: 228 to 332. Embodiment 52. The sgRNA comprising any one nucleic acid of SEQ ID NOs: 235 to 240, 265 to 285, and 309 to 329. 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 any one of the nucleic acids of array numbers 235 - 240, 265 - 285, and 309 - 329, and having a modification pattern identical to the modification pattern shown in the reference sequence identifier. Embodiment 54. The sgRNA of any one of Embodiments 51 - 53, further comprising three phosphorothioate (PS) bonds linking the first 4 nucleotides at the 5' end and three PS bonds linking the last 4 nucleotides at the 3' end.

[0130] Composition of B.dgRNA In some embodiments, the compositions and methods of the invention include a gRNA comprising a crRNA and a trRNA that direct a nuclease, such as Cas9, to a target DNA sequence. In some embodiments, the gRNAs are associated but on two separate RNA molecules (dual guide RNA or dgRNA).

[0131] Table 2 and Figure 21C provide an explanation of the domains of the crRNA used herein. The 5' terminal region may include a spacer region at or near the 5' end of the crRNA and may function to direct Cas9, for example, to a target region in the DNA described herein. In Table 2, "n" between regions represents a variable number, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, n is equal to 0. Any dgRNA described herein may include "n" between any domains.

[0132] Table 3 and FIG. 21C provide an explanation of the domains of the trRNA used herein. In Table 3, "n" between regions represents a variable number, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, n is equal to 0. Any dgRNA described herein may include "n" between any domains.

[0133] 1. Domains of dgRNA As described in Briner 2014, dgRNA can be developed based on specific functional domains herein referred to as "domains", such as a spacer domain involved in targeting, a lower stem domain, a bulge domain, an upper stem domain, a nexus domain, and a hairpin domain. In dgRNA, crRNA contains some components of gRNA, and trRNA contains some components of gRNA.

[0134] The regions of crRNA are provided in Table 2 and FIG. 21C. The regions of trRNA are provided in Table 3 and FIG. 21C. FIG. 21C shows a schematic diagram of an exemplary dgRNA. [Table 3] [Table 4]

[0135] a) 5’ terminal region In some embodiments, the dgRNA includes nucleotides at the 5' termini of the crRNA and trRNA, as shown in Tables 2-3 and FIG. 21C.

[0136] In some embodiments, the 5’ end of the crRNA comprises a spacer or guide region that functions to direct 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 do not function to direct the Cas protein to the target nucleotide region.

[0137] 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 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 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.

[0138] 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.

[0139] In some embodiments, the target sequence in the target gene 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 target gene 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 of the target gene may be 100% complementary or identical. In other embodiments, the guide region of the crRNA and the target region of the target gene may contain at least one mismatch. For example, when the full length of the target sequence is at least about 17, 18, 19, 20 or more base pairs, the guide region of the crRNA and the target region of the target gene may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, when the guide sequence contains at least about 17, 18, 19, 20 or more nucleotides, the guide region of the crRNA and the target region of the target gene may contain 1 to 6 mismatches. In some embodiments, when the guide sequence contains about 20 nucleotides, the guide region of the crRNA and the target region of the target gene may contain 1, 2, 3, 4, 5, or 6 mismatches.

[0140] In some embodiments, the trRNA includes a 5' end. In some embodiments, the trRNA includes a 5' end that partially forms the upper stem of the dgRNA. The 5' end of the trRNA is not complementary to the region of the target gene.

[0141] b) Lower stem In some embodiments, the dgRNA comprises a lower stem (LS) region. The lower stem region comprises a crRNA lower stem region and a trRNA lower stem region that associate as depicted in Figure 21C. In some embodiments, the lower stem region of the crRNA is at least partially complementary to the lower stem region of the trRNA. In some embodiments, the lower stem region of the crRNA is fully complementary to the lower stem region of the trRNA.

[0142] In some embodiments, the lower stem regions of the crRNA and the trRNA each comprise 6 nucleotides. In some embodiments, the lower stem regions of the crRNA and the trRNA each comprise fewer nucleotides than shown in Tables 2 and 3 and Figure 21C. In some embodiments, the lower stem region comprises more nucleotides than shown in Tables 2 and 3 and Figure 21C. When the lower stem region comprises fewer or more nucleotides than shown in the schematics of Tables 2 and 3 and Figure 21C, the modification pattern is maintained as will be apparent to those skilled in the art. In some embodiments, the number of nucleotides in the lower stem of the crRNA is different from the number of nucleotides in the lower stem of the trRNA.

[0143] c) Bulge In some embodiments, the dgRNA comprises a bulge (B) region. In some embodiments, the crRNA comprises one bulge region and the trRNA comprises one bulge region. In some embodiments, each bulge region comprises 1 to 4 nucleotides. In some embodiments, the bulge region of the crRNA comprises 2 nucleotides and the bulge region of the trRNA comprises 4 nucleotides.

[0144] In some embodiments, the bulge region of the crRNA is located between the lower stem region and the upper stem region of the crRNA. In some embodiments, the bulge region of the crRNA comprises 2 nucleotides. In some embodiments, the bulge region of the crRNA comprises nucleotides B1 and B2 as shown in Tables 2 and Figure 21C.

[0145] In some embodiments, the bulge region of the trRNA is located between the upper stem region and the lower stem region of the trRNA. In some embodiments, the bulge region of the trRNA comprises 4 nucleotides. In some embodiments, the bulge region of the trRNA comprises nucleotides B1 to B4, as shown in Table 3 and FIG. 21C.

[0146] In some embodiments, the presence of the bulge results in a directional kink between the upper stem module and the lower stem module in the dgRNA. The bulge of the crRNA and the bulge of the trRNA may be partially complementary. The bulge of the crRNA and the bulge of the trRNA may not have complementarity.

[0147] In some embodiments, the bulge regions of the crRNA and the trRNA comprise more nucleotides than shown in Table 2 and Table 3 and FIG. 21C. If the bulge region comprises fewer or more nucleotides than shown in the schematics of Table 2 and Table 3 and FIG. 21C, the modification pattern is maintained, as will be apparent to those skilled in the art. In some embodiments, the number of nucleotides in the bulge of the crRNA is different from the number of nucleotides in the bulge of the trRNA.

[0148] d) Upper stem In some embodiments, the dgRNA comprises an upper stem (US) region. The upper stem region comprises the crRNA upper stem region and the trRNA upper stem region that associate as depicted in FIG. 21C. In some embodiments, the upper stem region of the crRNA is at least partially complementary to the upper stem region of the trRNA. In some embodiments, the upper stem region of the crRNA is fully complementary to the upper stem region of the trRNA.

[0149] In some embodiments, the upper stem region of the crRNA comprises 14 nucleotides. In some embodiments, the upper stem region of the trRNA comprises 11 nucleotides. In some embodiments, the upper stem regions of the crRNA and trRNA each comprise fewer nucleotides than shown in Tables 2 and 3 and FIG. 21C. In some embodiments, the upper stem regions of the crRNA and trRNA each comprise more nucleotides than shown in Tables 2 and 3 and FIG. 21C. When the upper stem region comprises fewer or more nucleotides than shown in the schematics of Tables 2 and 3 and FIG. 21C, the modification pattern is maintained, as will be apparent to those skilled in the art.

[0150] In some embodiments, the upper stem of the crRNA comprises nucleotides US1 to US14, as shown in Tables 2 and 21C.

[0151] In some embodiments, the upper stem of the trRNA comprises nucleotides US1 to US11, as shown in Tables 3 and 21C.

[0152] e) Nexus In some embodiments, the dgRNA comprises a trRNA containing a nexus region. In some embodiments, the nexus is between the lower stem region and the hairpin 1 region of the trRNA. In some embodiments, the nexus is located immediately downstream of the lower stem of the trRNA. In some embodiments, the nexus comprises 18 nucleotides. In some embodiments, the nexus region of the trRNA comprises nucleotides N1 to N18, as shown in Tables 3 and 21C. In some embodiments, the nexus comprises fewer nucleotides than shown in Tables 3 and 21C. In some embodiments, the nexus region of the trRNA comprises more nucleotides than shown in Tables 3 and 21C. When the nexus region comprises fewer or more nucleotides than shown in Tables 3 and 21C, the modification pattern is maintained, as will be apparent to those skilled in the art.

[0153] In some embodiments, the nexus region has nucleotides for which the nucleic acid sequences are complementary when read in opposite directions. In some embodiments, the complementarity in the nucleic acid sequence leads to the secondary structure of the stem and / or stem-loop in the sgRNA (e.g., certain nucleotides in the nexus region may base pair with each other). In some embodiments, the nexus region may not be completely complementary to each other when read in opposite directions.

[0154] f) Hairpin In some embodiments, the hairpin region of the trRNA is downstream of the nexus region. In some embodiments, the region of nucleotides immediately downstream of the nexus region is referred to as "hairpin 1". In some embodiments, the region of nucleotides immediately downstream of the hairpin 1 region is referred to as "hairpin 2". In some embodiments, the hairpin region includes hairpin 1 and hairpin 2. In some cases, hairpin 1 and hairpin 2 are separated by one or more nucleotides "n". In some embodiments, n = 1. In some embodiments, the trRNA includes only hairpin 1 or hairpin 2.

[0155] Substitution of the hairpin 1 region of the trRNA by two nucleotides has been shown to enable the editing activity of the Cas RNP (see US Patent Publication No. 20150376586, Figure 16). In some embodiments, the trRNA includes substitution of hairpin 1 by nucleotide "n", where "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 by two nucleotides.

[0156] In some embodiments, the hairpin 1 of the trRNA includes 12 nucleotides immediately downstream of the nexus region. In some embodiments, the hairpin 1 region of the trRNA includes nucleotides H1-1 to H1-12 as shown in Table 3 and Figure 21C.

[0157] In some embodiments, the non-hairpin nucleotides are present between the hairpin 1 region and the hairpin 2 region of the trRNA. In some embodiments, 1 to 2 non-hairpin nucleotides are present between hairpin 1 and hairpin 2.

[0158] In some embodiments, hairpin 2 of the trRNA contains 15 nucleotides after (3’) hairpin 1. In some embodiments, the hairpin 2 region of the trRNA contains nucleotides H2-1 to H2-15, as shown in Table 3 and FIG. 21C. In some embodiments, the hairpin 2 region of the trRNA contains nucleotides H2-1 to H2-15, as shown in Table 3, and “n” between hairpin 1 and hairpin 2 is 1 or 2.

[0159] In some embodiments, the hairpin region of the trRNA contains more nucleotides than shown in Table 3 and FIG. 21C. If the hairpin region contains fewer or more nucleotides than shown in Table 3 and FIG. 21C, the modification pattern is maintained, as will be apparent to those skilled in the art.

[0160] In some embodiments, the hairpin region has nucleotides whose nucleic acid sequences are complementary when read in the opposite direction. In some embodiments, the hairpin regions may not be completely complementary to each other when read in the opposite direction (e.g., the top or loop of the hairpin contains non-pairing nucleotides).

[0161] In some embodiments, the trRNA contains a substitution of hairpin 1 by nucleotide “n”, where “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 by 2 nucleotides.

[0162] g) 3’ terminus In some embodiments, the dgRNA comprises a trRNA that includes a 3' terminal region containing additional nucleotides after (3') the hairpin region(s). In some embodiments, the 3' terminal region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments, the 3' terminal region comprises 1, 2, 3, or 4 nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments, the 3' terminal region comprises 4 nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments, the 3' terminal region comprises 1, 2, or 3 nucleotides that do not associate with the secondary structure of the hairpin.

[0163] 2. Modification of dgRNA 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, both the crRNA and trRNA of the dgRNA comprise modifications.

[0164] In some embodiments, the gRNA described herein results in two separate RNA molecules (dual guide or dgRNA). See Table 2, Table 3, and Figure 21C.

[0165] In some embodiments, the invention comprises a dgRNA comprising or consisting of a) any one of the crRNA sequences of SEQ ID NOs: 1-187; and b) any one of the trRNA sequences set forth in SEQ ID NOs: 188-227.

[0166] In some embodiments, a dgRNA is provided that comprises any one of the modified crRNA sequences of 1-187.

[0167] In some embodiments, a dgRNA is provided that comprises any one of the modified trRNA sequences of 188-227.

[0168] In some embodiments, a dgRNA is provided that includes any one of the modified crRNA sequences of SEQ ID NOs: 19-31, 53-73, and 104-130. In some embodiments, the invention includes a dgRNA that includes any one of the modified sequences of SEQ ID NOs: 19-31, 53-73, and 104-130, wherein the crRNA is at least partially complementary to a target sequence and further includes a 5' spacer sequence that directs Cas9 to its target for cleavage.

[0169] In some embodiments, the invention includes a crRNA that includes any one of the sequences set forth in SEQ ID NOs: 1-187. In some embodiments, the invention includes a crRNA that includes or consists of any one of the sequences set forth in SEQ ID NOs: 19-31, 53-73, and 104-130. In some embodiments, the invention includes a crRNA that includes any one of the sequences set forth in SEQ ID NOs: 19-31, 53-73, and 104-130 and a spacer region.

[0170] In some embodiments, the invention includes a trRNA that includes or consists of any one of the sequences set forth in SEQ ID NOs: 188-277.

[0171] In some embodiments, the invention includes a crRNA that includes nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one nucleotide of SEQ ID NOs: 1-187, 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 be 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% different compared to those shown in the sequence, but the modifications remain unchanged.

[0172] In some embodiments, the present invention includes a trRNA comprising nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of nucleotides 188 to 277, and the modification pattern is the same as the modification pattern shown by the reference sequence identifier. That is, nucleotides A, U, C, and G may be 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% different compared to those shown in the sequence, but the modifications on each nucleotide remain unchanged.

[0173] 3. crRNA, trRNA, and dgRNA with modifications In some embodiments, the crRNA includes one or more modified nucleotides within one or more of the 5'-end, lower stem, bulge, upper stem, and 3'-end.

[0174] In some embodiments, the modification includes 2'-O-Me.

[0175] In some embodiments, the modification includes 2'-F.

[0176] In some embodiments, the modification includes phosphorothioate (PS) bonds that link one or more nucleotides. In some embodiments, the modification is three PS bonds that link the first four nucleotides at the 5'-end and three PS bonds that link the last four nucleotides at the 3'-end.

[0177] In some embodiments, the modification includes inverted abasic nucleotides.

[0178] In some embodiments, a crRNA is provided that includes 2'-O-Me modified nucleotides at each nucleotide in the upper stem. 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.

[0179] In some embodiments, a crRNA is provided that includes 2'-O-Me modified nucleotides at each nucleotide in the upper stem, and at LS1 and LS6 in the lower stem. In some embodiments, the crRNA further includes one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as during 5' and / or 3' end modifications.

[0180] In some embodiments, a crRNA is provided that includes 2'-O-Me modified nucleotides at each nucleotide in the upper stem, and at LS1, LS5, and LS6 in the lower stem. In some embodiments, the crRNA further includes one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as during 5' and / or 3' end modifications.

[0181] In some embodiments, the present invention includes a crRNA that includes 2'-F modified nucleotides at LS1, LS2, and LS6 in the lower stem. In some embodiments, the crRNA further includes 2'-F modified nucleotides at each of B1 and B2 in the bulge region. In some embodiments, the present invention includes a crRNA that includes 2'-F modified nucleotides at LS1, LS2, and LS6 in the lower stem, and at each of B1 and B2 in the bulge region. In some embodiments, the crRNA further includes one or more 2'-O-Me or 2'-O-moe modified nucleotides in the 5' and / or 3' terminal regions, such as during 5' and / or 3' end modifications.

[0182] In some embodiments, the crRNA comprises 2'-O-Me modified nucleotides in nucleotides LS1 and LS6 in the lower stem region; each of the nucleic acids in the bulge region; and 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 not modified. 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, for example, during 5' and / or 3' end modifications.

[0183] In some embodiments, the crRNA comprises 2'-fluoro (2'-F) modified nucleotides in LS1, LS2, and LS6 in the lower stem region and each of the nucleotides in the bulge region. In some embodiments, the crRNA comprises 2'-fluoro (2'-F) modified nucleotides in LS1, LS2, and LS6 in the lower stem region and B2 in B2 and the bulge region. In some embodiments, the crRNA comprises 2'-fluoro (2'-F) modified nucleotides in LS1-LS6 in the lower stem region and each of the nucleotides in the bulge 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, for example, during 5' and / or 3' end modifications.

[0184] In some embodiments, the present invention comprises a trRNA comprising one or more modified nucleotides within one or more of the following regions: the 5' end, the upper stem region; the bulge region; the lower stem region; the nexus region; the hairpin 1 region; the intervening region between the hairpin 1 region and the hairpin 2 region; the hairpin 2 region; and the 3' end region.

[0185] In some embodiments, the modification comprises 2'-O-Me.

[0186] In some embodiments, the modification comprises 2'-F.

[0187] In some embodiments, the modification comprises phosphorothioate (PS) linkages that link one or more nucleotides. In some embodiments, the modification is three PS linkages that link the first four nucleotides at the 5' end and three PS linkages that link the last four nucleotides at the 3' end.

[0188] In some embodiments, the modification comprises inverted abasic nucleotides.

[0189] In some embodiments, the trRNA comprises 2'-O-Me modified nucleotides at each nucleic acid in the upper stem; B1 and B2 in the bulge region; LS1 and LS2 in the lower stem region; N3, N4, N5, N15, N16, N17, and N18 in the nexus 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, for example, during 5' and / or 3' terminal modifications.

[0190] In some embodiments, the trRNA comprises 2'-O-Me modified nucleotides at each nucleic acid in the upper stem; each nucleotide in the bulge region; LS1, LS2, LS5, and LS6 in the lower stem region; N3 - N5, N10 - N18 in the nexus 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, for example, during 5' and / or 3' terminal modifications.

[0191] In some embodiments, the trRNA contains 2'-F modified nucleotides at N15 to N18 in the nexus region. In some embodiments, the trRNA further contains one or more 2'-F modified nucleotides in the 5' and / or 3' terminal regions, such as during 5' and / or 3' terminal modification.

[0192] In some embodiments, the trRNA contains 2'-F modified nucleotides at LS4 and LS5 in the lower stem region and at N13 - N18 in the nexus region. In some embodiments, the trRNA further contains one or more 2'-F modified nucleotides in the 5' and / or 3' terminal regions, such as during 5' and / or 3' terminal modification.

[0193] In some embodiments, the trRNA contains 2'-F modified nucleotides at LS1, LS3, and LS5 in the lower stem and 2'-O-Me modified nucleotides at LS2, LS4, and LS6 in the lower stem.

[0194] In some embodiments, a CRISPR RNA (crRNA) comprising one or more modifications within one or more of the following regions: the first 5 nucleotides at the 5' end; the lower stem region; the bulge region; the upper stem region; and the last 5 nucleotides at the 3' end is disclosed herein. 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 3 nucleotides at the 5' end and the last 3 nucleotides at the 3' end are modified. In some embodiments, the first 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked by 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 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked by a PS bond and the first 3 nucleotides at the 5' end and the last 3 nucleotides at the 3' end comprise a 2'-O-Me modification. In some embodiments, the first 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked by a PS bond and the first 3 nucleotides at the 5' end and the last 3 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.

[0195] In some embodiments, the present invention includes a crispr RNA (crRNA) comprising the following nucleotides: LS1 and LS6 in the lower stem region; and 2'-O-Me modified nucleic acids at each nucleotide in the upper stem region. In some embodiments, the crRNA further comprises three phosphorothioate (PS) linkages that link the first four nucleotides at the 5' end and three PS linkages that link the last four nucleotides at the 3' end. In some embodiments, the crRNA further comprises 2'-O-Me or 2'-F modified nucleic acids 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, LS1, LS2, and LS6 are modified with 2'-F. In some embodiments, each nucleotide in the bulge region is modified with 2'-F.

[0196] In some embodiments, a crispr RNA (crRNA) is disclosed herein that comprises 2'-F modified nucleic acids at the following nucleotides: LS1, LS2, and LS6 in the lower stem region and each nucleotide in the bulge region. In some embodiments, the crRNA further comprises three phosphorothioate (PS) linkages that link the first four nucleotides at the 5' end and three PS linkages that link the last four nucleotides at the 3' end. In some embodiments, the crRNA further comprises 2'-O-Me or 2'-F modified nucleic acids 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.

[0197] In some embodiments, a crRNA comprising any one of the nucleic acids of SEQ ID NOs: 1-187 is provided. In some embodiments, a crRNA comprising any one of the nucleic acids of SEQ ID NOs: 19-31, 53-73, 104-130, and 161-187 is provided. 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 any one of the nucleic acids of SEQ ID NOs: 19-31, 53-73, 104-130, and 161-187, and 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) linkages that link the first four nucleotides at the 5' end and three PS linkages that link the last four nucleotides at the 3' end.

[0198] Also included are tracr RNAs (trRNAs) that contain one or more modifications within one or more of the following regions: the first 5 nucleotides at the 5' end; the upper stem region; the bulge region; the lower stem region; the nexus region; the hairpin 1 region; the hairpin 2 region; and the last 5 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 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked by a phosphorothioate (PS) bond. In some embodiments, the first 3 nucleotides at the 5' end and the last 3 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 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked by a PS bond and the first 3 nucleotides at the 5' end and the last 3 nucleotides at the 3' end comprise a 2'-O-Me modification. In some embodiments, the first 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked by a PS bond and the first 3 nucleotides at the 5' end and the last 3 nucleotides at the 3' end comprise a 2'-F modification. In some embodiments, each nucleotide in the upper stem region is modified with 2'-O-Me. In some embodiments, B1 and B2 within the bulge region are modified with 2'-O-Me. In some embodiments, N3, N4, N5, N15, N16, N17, and N18 within the nexus 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.

[0199] In some embodiments, the invention includes a tracrRNA (trRNA) comprising 2'-O-Me modified nucleic acids at the following nucleotides: each nucleotide in the upper stem; B1 and B2 in the bulge region; N3, N4, N5, N15, N16, N17, and N18 in the nexus region; each nucleotide in the hairpin 1 region; and each nucleotide in the hairpin 2 region. In some embodiments, the trRNA further comprises three phosphorothioate (PS) linkages that link the first four nucleotides at the 5' terminus and three PS linkages that link the last four nucleotides at the 3' terminus. In some embodiments, the trRNA further comprises a 2'-O-Me or 2'-F modified nucleotide at the first three nucleotides at the 5' terminus and a 2'-O-Me or 2'-F modified nucleic acid at the last three nucleotides at the 3' terminus. 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) linkages that link the first four nucleotides at the 5' terminus and three PS linkages that link the last four nucleotides at the 3' terminus. 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' terminus and a 2'-O-Me or 2'-F modified nucleic acid at the last three nucleotides at the 3' terminus.

[0200] In some embodiments, a trRNA comprising any one of the nucleic acids of SEQ ID NOs: 188 to 227 is provided. In some embodiments, a trRNA 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 any one of the nucleic acids of SEQ ID NOs: 188 to 227, and 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 linking the first four nucleotides at the 5' end and three PS bonds linking the last four nucleotides at the 3' end.

[0201] In some cases, a dual guide comprising a crRNA and a trRNA is provided, wherein the crRNA comprises any one of the nucleic acids of SEQ ID NOs: 1 to 187 and the trRNA comprises any one of the nucleic acids of SEQ ID NOs: 188 to 227.

[0202] Included are dual guides comprising a crRNA disclosed herein and a trRNA disclosed herein, and also 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.

[0203] In some embodiments, the following are included: Embodiment 55. A crispr RNA (crRNA) comprising one or more modifications within one or more of the following regions: a. The first five nucleotides at the 5' end, b. The lower stem region, c. The bulge region, d. The upper stem region, e. The last five nucleotides at the 3' end. Embodiment 56. The crRNA of embodiment 55, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. Embodiment 57. The crRNA of embodiment 55, wherein the modification comprises a 2'-fluoro (2'-F) modified nucleotide. Embodiment 58. The crRNA of embodiment 55, wherein the modification comprises a phosphorothioate (PS) bond between nucleotides. Embodiment 59. The crRNA of any one of embodiments 55-58, wherein the first 3 nucleotides at the 5' terminus and the last 3 nucleotides at the 3' terminus are modified. Embodiment 60. The crRNA of any one of embodiments 55-58, wherein the first 4 nucleotides at the 5' terminus and the last 4 nucleotides at the 3' terminus are linked by a phosphorothioate (PS) bond. Embodiment 61. The crRNA of embodiment 59, wherein the modification comprises 2'-O-Me. Embodiment 62. The crRNA of embodiment 59, wherein the modification comprises 2'-F. Embodiment 63. The crRNA of any one of embodiments 55-62, wherein the first 4 nucleotides at the 5' terminus and the last 4 nucleotides at the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' terminus and the last 3 nucleotides at the 3' terminus comprise a 2'-O-Me modification. Embodiment 64. The crRNA of any one of embodiments 55-62, wherein the first 4 nucleotides at the 5' terminus and the last 4 nucleotides at the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' terminus and the last 3 nucleotides at the 3' terminus comprise a 2'-F modification. Embodiment 65. The crRNA of any one of embodiments 55-60, wherein LS1 and LS6 are modified with 2'-O-Me. Embodiment 66. A crRNA of any one of embodiments 55-60 and 65, wherein each nucleotide in the upper stem region is modified with 2'-O-Me. Embodiment 67. A crispr RNA (crRNA), a. LS1 and LS6 in the lower stem region, and b. each nucleotide in the upper stem region, the crRNA containing 2'-O-Me modified nucleotides. 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. 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. Embodiment 70. A crRNA of any one of embodiments 55-60, wherein LS1, LS2, and LS6 are modified with 2'-F. Embodiment 71. A crRNA of any one of embodiments 55-60 and 70, wherein each nucleotide in the bulge region is modified with 2'-F. Embodiment 72. A crispr RNA (crRNA), a. LS1, LS2, and LS6 in the lower stem region, and b. each nucleotide in the bulge region, the crRNA containing 2'-F modified nucleotides. Embodiment 73. The crRNA of any one of embodiments 70-72, 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. Embodiment 74. The crRNA of embodiment 72 or 73, further comprising a 2'-O-Me or 2'-F modified nucleotide at the first 3 nucleotides at the 5' end and a 2'-O-Me or 2'-F modified nucleotide at the last 3 nucleotides at the 3' end. Embodiment 75. A crRNA comprising any one nucleic acid of SEQ ID NOs: 1 to 187. Embodiment 76. A crRNA comprising any one nucleic acid of SEQ ID NOs: 19 to 31, 53 to 73, 104 to 130, and 161 to 187. 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 any one nucleic acid of SEQ ID NOs: 19 to 31, 53 to 73, 104 to 130, and 161 to 187, and having a modification pattern identical to the modification pattern shown in the reference sequence identifier. Embodiment 78. The crRNA of any one of embodiments 75 to 77, further comprising three phosphorothioate (PS) bonds linking the first 4 nucleotides at the 5' end and three PS bonds linking the last 4 nucleotides at the 3' end. Embodiment 79. A tracr RNA (trRNA), Comprising the following regions: a. The first 5 nucleotides at the 5' end, b. The upper stem region, c. The bulge region, d. The lower stem region, e. The nexus region, f. The hairpin 1 region, g. The hairpin 2 region, h. The last 5 nucleotides at the 3' end, and comprising one or more modifications within one or more of them. Embodiment 80. The tRNA of embodiment 79, wherein the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. Embodiment 81. The tRNA of embodiment 79, wherein the modification comprises a 2'-fluoro (2'-F) modified nucleotide. Embodiment 82. The tRNA of embodiment 79, wherein the modification comprises a phosphorothioate (PS) bond between nucleotides. Embodiment 83. The tRNA of any one of embodiments 79 to 82, wherein the first 4 nucleotides at the 5' terminus and the last 4 nucleotides at the 3' terminus are linked by a phosphorothioate (PS) bond. Embodiment 84. The tRNA of any one of embodiments 79 to 82, wherein the first 3 nucleotides at the 5' terminus and the last 3 nucleotides at the 3' terminus are modified. Embodiment 85. The tRNA of embodiment 84, wherein the modification comprises 2'-O-Me. Embodiment 86. The tRNA of embodiment 84, wherein the modification comprises 2'-F. Embodiment 87. The tRNA of any one of embodiments 79 to 86, wherein the first 4 nucleotides at the 5' terminus and the last 4 nucleotides at the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' terminus and the last 3 nucleotides at the 3' terminus comprise a 2'-O-Me modification. Embodiment 88. The tRNA of any one of embodiments 79 to 86, wherein the first 4 nucleotides at the 5' terminus and the last 4 nucleotides at the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' terminus and the last 3 nucleotides at the 3' terminus comprise a 2'-F modification. Embodiment 89. The tRNA of any one of embodiments 79 to 84, wherein each nucleotide in the upper stem region is modified with 2'-O-Me. Embodiment 90. A trRNA according to any one of Embodiments 79 to 84 and 89, wherein B1 and B2 in the bulge region are modified with 2'-O-Me. Embodiment 91. A trRNA according to any one of Embodiments 79 to 84 and 89 to 90, wherein N3, N4, N5, N15, N16, N17, and N18 in the nexus region are modified with 2'-O-Me. Embodiment 92. A trRNA according to 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. Embodiment 93. A trRNA according to 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. Embodiment 94. A tracr RNA (trRNA) comprising: a. each nucleotide in the upper stem; b. B1 and B2 in the bulge region; c. N3, N4, N5, N15, N16, N17, and N18 in the nexus region; d. each nucleotide in the hairpin 1 region; e. each nucleotide in the hairpin 2 region, and containing 2'-O-Me modified nucleotides. 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. Embodiment 96. The crRNA of Embodiment 94 or 95, further comprising a 2'-O-Me or 2'-F modified nucleotide 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. Embodiment 97. The tRNA of any one of Embodiments 79-84, wherein N15, N16, N17, and N18 are modified with 2'-F. Embodiment 98. LS1, LS3, and LS5 are modified with 2'-F, and LS2, LS4, and LS6 are modified with 2'-O-Me. The tRNA of any one of Embodiments 79-84 and 97. Embodiment 99. The tRNA of any one of Embodiments 87-98, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end and three PS bonds connecting the last 4 nucleotides at the 3' end. Embodiment 100. The tRNA of Embodiment 98 or 99, further comprising a 2'-O-Me or 2'-F modified nucleotide at the first 3 nucleotides at the 5' end and a 2'-O-Me or 2'-F modified nucleotide at the last 3 nucleotides at the 3' end. Embodiment 101. The tRNA comprising any one of the nucleic acids of SEQ ID NOs: 188-227. Embodiment 102. The tRNA 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 the nucleic acids of SEQ ID NOs: 188-227, and the modification pattern is the same as the modification pattern shown in the reference sequence identifier. Embodiment 103. The tRNA of any one of Embodiments 101-102, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end and three PS bonds connecting the last 4 nucleotides at the 3' end. Embodiment 104. A dual guide comprising crRNA and trRNA, wherein the crRNA contains any one nucleotide of SEQ ID NOs: 1 to 187, and the trRNA contains any one nucleic acid of SEQ ID NOs: 188 to 227. Embodiment 105. A dual guide comprising any one crRNA of Embodiments 55 to 78 and any one trRNA of Embodiments 79 to 103. Embodiment 106. A dual guide comprising any one crRNA of Embodiments 55 to 78 and an unmodified trRNA. Embodiment 107. A dual guide comprising an unmodified crRNA and any one trRNA of Embodiments 79 to 103.

[0204] C. Modification of Terminal Nucleotides In some embodiments, the nucleotides at the 5' or 3' terminus of any of the guide RNAs described herein are modified. In some embodiments, for example, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region of a guide RNA, such as 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 a guide RNA contain more than one modification. In some embodiments, at least 1 of the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in the 3' terminal region is modified. In some embodiments, at least 2 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 3 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 includes a PS linkage.

[0205] In some embodiments, the 5' end of the 5' terminal region is modified. For example, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides of sgRNA, dgRNA, crRNA, trRNA, or both 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 contain more than one modification. In some embodiments, at least one of the 1, 2, 3, 4, 5, 6, or 7 nucleotides at the terminus (i.e., the first) at the 5' end is modified. In some embodiments, at least two of the 1, 2, 3, 4, 5, 6, or 7 nucleotides at the terminus at the 5' end are modified. In some embodiments, at least three of the 1, 2, 3, 4, 5, 6, or 7 nucleotides at the terminus at the 5' end are modified. In some embodiments, the modification includes a PS linkage.

[0206] In some embodiments, both the 5' end and the 3' end (e.g., the terminus) of the guide RNA, such as sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, are modified. In some embodiments, only the 5' end of the guide RNA, such as sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, is modified. In some embodiments, only the 3' end of the guide RNA, such as sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, is modified.

[0207] In some embodiments, the gRNA comprises a modification at one, two, three, four, five, six, or seven of the first seven nucleotides at the 5' end of the gRNA. In some embodiments, the gRNA comprises a modification at one, two, three, four, five, six, or seven of the seven terminal nucleotides at the 3' end. In some embodiments, two, three, or four of the first four nucleotides at the 5' end and / or two, three, or four of the four terminal nucleotides at the 3' end are modified. In some embodiments, two, three, or four of the first four nucleotides at the 5' end are linked by phosphorothioate (PS) bonds.

[0208] In some embodiments, the 5' and / or 3' end modifications comprise 2'-O-methyl (2'-O-Me) or 2'-O-(2-methoxyethyl) (2'-O-moe) modifications to the nucleotides. In some embodiments, the modification comprises 2'-fluoro (2'-F) modification to the nucleotides. In some embodiments, the modification comprises phosphorothioate (PS) linkages between nucleotides. In some embodiments, the modification comprises inverted abasic nucleotides. In some embodiments, the modification comprises more than one modification selected from 2'-O-Me, 2'-O-moe, 2'-fluoro (2'-F), phosphorothioate (PS) linkages between nucleotides, and inverted abasic nucleotides. In some embodiments, equivalent modifications are included.

[0209] In some embodiments, a guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, comprises one or more phosphorothioate (PS) linkages between the first 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5' end. In some embodiments, a guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, comprises one or more PS linkages between the last 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 3' end. In some embodiments, a guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, comprises one or more PS linkages between the last 1, 2, 3, 4, 5, 6, or 7 nucleotides at both the 5' and 3' ends. In some embodiments, in addition to the PS linkages, the 5' and 3' terminal nucleotides may comprise 2'-O-Me, 2'-O-moe, or 2'-F modified nucleotides.

[0210] In some embodiments, a guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA, comprises modified nucleotides at the 5' and 3' ends, as well as modified nucleotides in one or more other regions described in Tables 1-3 and Figures 21A or 21C.

[0211] In some embodiments, crRNA, trRNA, or both crRNA and trRNA comprise modified nucleotides that are not at the 5' or 3' termini. Specific patterns of modification are described below and in Table 4.

[0212] 3. Delivery of gRNA and Cas Protein In some embodiments, in addition to at least one gRNA, the compositions provided herein further comprise a nuclease. In some embodiments, the nuclease is a Cas protein. In some embodiments, the gRNA in combination with the Cas protein is referred to as a Cas RNP. 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 wild-type Cas9. In some embodiments, the Cas9 protein is derived from the Cas9 protein of Streptococcus pyogenes, e.g., Cas9 of S. pyogenes. In some embodiments, the Cas9 protein is not derived from S. pyogenes but functions in the same manner as Cas9 of S. pyogenes, and the gRNA specific for Cas9 of S. pyogenes directs the non-S. pyogenes Cas9 to its target site. In some embodiments, the Cas induces a double-strand break in the target DNA. Equivalents of the Cas9 protein of S. pyogenes are encompassed by the embodiments described herein.

[0213] Cas9 includes its modifications and variants. A modified version of Cas9 having an inactive catalytic domain of either RuvC or HNH is referred to as a "nickase". A nickase cleaves only one strand of the target DNA, thus creating a single-strand break. A single-strand break may also be known 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.

[0214] In some embodiments, the Cas protein may be modified to contain only one functional nuclease domain. For example, the Cas protein may be modified by mutating or completely or partially deleting one of the nuclease domains to reduce its nucleic acid cleavage activity. In some embodiments, a nickase Cas having a reduced activity RuvC domain is used. In some embodiments, a nickase Cas having an inactive RuvC domain is used. In some embodiments, a nickase Cas having a reduced activity HNH domain is used. In some embodiments, a nickase Cas having an inactive HNH domain is used.

[0215] In some embodiments, the conserved amino acids within the nuclease domain of the Cas protein are substituted to reduce or alter the nuclease activity. In some embodiments, the Cas protein may contain amino acid substitutions in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the Cas9 protein of S. Pyogenes). In some embodiments, the Cas protein may contain amino acid substitutions in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the Cas9 protein of S. Pyogenes).

[0216] In some embodiments, the RNP complex described herein comprises a nickase and a pair of guide RNAs that are complementary to the sense and antisense strands of a target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to the target sequence and introduce a double-strand break (DSB) by generating a nick on the opposite strand of the target sequence (i.e., double nick formation). In some embodiments, the use of double nick formation can improve specificity and reduce off-target effects. In some embodiments, the nickase Cas is used with two separate guide RNAs that target opposite strands of DNA to generate a double nick in the target DNA. In some embodiments, the nickase Cas is used with two separate guide RNAs selected to be in proximity to generate a double nick in the target DNA.

[0217] In some embodiments, a chimeric Cas protein in which one domain or region of the protein is replaced by a portion of a different protein is used. In some embodiments, the Cas nuclease domain may be replaced by a domain from a different nuclease, such as Fok1. In some embodiments, the Cas protein may be a modified nuclease.

[0218] In some embodiments, the Cas protein comprises a fusion protein comprising catalytically inactive Cas9 linked to a heterologous functional domain (see, e.g., International Patent Publication No. WO 2014 / 152432). In some embodiments, the catalytically inactive Cas9 is from S. Pyogenes. In some embodiments, the catalytically inactive Cas9 comprises mutations that inactivate 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.

[0219] A.PAM In some embodiments, the target sequence may be adjacent to the PAM. In some embodiments, the PAM may be adjacent to or within one, two, three, or four nucleotides at the 3’ end of the target sequence. The length and sequence of the PAM may depend on the Cas protein being used. For example, the PAM may be selected from the consensus sequence of a particular Cas9 protein or Cas9 ortholog or a particular PAM sequence, including those disclosed in FIG. 1 of Ran et al., Nature 520: 186-191 (2015), which is incorporated herein by reference. In some embodiments, the PAM may include a length of two, three, four, five, six, seven, eight, nine, or ten nucleotides. Non-limiting exemplary PAM sequences include NGG, NAG, NGA, NGAG, NGCG, NNGRRT, TTN, NGGNG, NG, NAAAAN, NNA AAW, NNNNACA, GNNNCNNA, and NNNNGATT, where N is defined as any nucleotide, W is defined as either A or T, and R is defined as either A or G. In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be NNA AAW.

[0220] B. Delivery of Modified gRNA Lipid nanoparticles (LNPs) are a well-known means for the delivery of nucleotides and protein cargos and may be used for the delivery of the gRNAs, mRNAs, Cas9, and RNPs disclosed herein. In some embodiments, the LNP delivers a nucleic acid, a protein, or a nucleic acid together with a protein.

[0221] In some embodiments, the invention includes a method of delivering any one of the gRNAs disclosed herein to a subject, wherein the gRNA is associated with an LNP. In some embodiments, the gRNA / LNP is also associated with Cas9 or mRNA encoding Cas9.

[0222] In some embodiments, the present invention comprises a composition comprising any one of the disclosed gRNAs and an LNP. In some embodiments, the composition further comprises Cas9 or mRNA encoding Cas9.

[0223] In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate). In some embodiments, the LNP comprises a molar ratio (N:P) of cationic lipid amine to RNA phosphate of about 4.5.

[0224] In some embodiments, the LNP associated with the gRNAs disclosed herein is for use in the preparation of a medicament for treating a disease or disorder.

[0225] Electroporation is a well-known means for cargo delivery and any electroporation methodology may be used for delivery of any one of the gRNAs disclosed herein. In some embodiments, electroporation may be used to deliver any one of the gRNAs disclosed herein and Cas9 or mRNA encoding Cas9.

[0226] In some embodiments, the present invention comprises a method of delivering any one of the gRNAs disclosed herein to ex vivo cells, wherein the gRNA is associated with an LNP or not associated with an LNP. In some embodiments, the gRNA / LNP or gRNA is also associated with Cas9 or mRNA encoding Cas9.

[0227] 4. Methods of Gene Modulation In some embodiments, the invention includes a pharmaceutical formulation comprising any one of the gRNAs disclosed herein together with a pharmaceutically acceptable carrier. In some embodiments, the invention includes a pharmaceutical formulation comprising any one of the gRNAs disclosed herein and an LNP together with a pharmaceutically acceptable carrier. In some embodiments, the invention includes a pharmaceutical formulation comprising any one of the gRNAs disclosed herein, a Cas9 protein or an mRNA encoding a Cas9 protein, and an LNP together with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation is for use in the preparation of a medicament for treating a disease or disorder. In some embodiments, the invention includes a method of treating a human patient comprising administering any one of the gRNAs or pharmaceutical formulations described herein.

[0228] In some embodiments, the invention includes 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.

[0229] In some embodiments, the invention includes a method or use for modulation of a target gene comprising administering or delivering a Cas protein or Cas mRNA and any one or more of the gRNAs disclosed herein. In some embodiments, the modulation is editing of the target gene. In some embodiments, the modulation is a change in the expression of a protein encoded by the target gene.

[0230] In some embodiments, the method or use results in gene editing. In some embodiments, the method or use results in a double-strand break in a target gene. In some embodiments, the method or use results in the formation of indel mutations during non-homologous end joining of DSBs. In some embodiments, the method or use results in an insertion or deletion of nucleotides in the target gene. In some embodiments, the insertion or deletion of nucleotides in the target gene leads to a frameshift mutation or premature stop codon that results in a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in the target gene leads to knockdown or elimination of target gene expression. In some embodiments, the method or use includes homologous recombination repair of DSBs. In some embodiments, the method or use further includes delivering a template to the cell, and at least a portion of the template is incorporated into the target DNA at or near the double-strand break site induced by the Cas protein.

[0231] In some embodiments, the method or use results in gene modulation. In some embodiments, gene modulation 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.

[0232] In some embodiments, any of the gRNAs disclosed herein may be useful in the preparation of an agent for treating a disease or disorder.

[0233] A. Measurement of Gene Modulation The effectiveness of the modified gRNA can be tested in vitro and in vivo. In some embodiments, the invention includes one or more of the gRNAs disclosed herein, which result in gene modulation when provided to cells together with Cas9. In some embodiments, the effectiveness of the gRNA can be measured in an in vitro or in vivo assay.

[0234] 1. In vitro measurement of Cas effectiveness In some embodiments, the activity of a CasRNP comprising a modified sgRNA is compared to the activity of a Cas RNP comprising an unmodified sgRNA.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] In some embodiments, the efficacy of a gRNA in increasing or decreasing the expression of a target protein is determined by measuring the amount of the target protein. In some embodiments, the present invention includes any one of the gRNAs described herein, and the gRNA increases or decreases the amount of protein produced from the targeted gene. In some embodiments, the present invention includes a method of modulating protein expression, comprising administering to a subject any one of the gRNAs disclosed herein, wherein the gRNA directs Cas9 to the gene encoding the target protein, and the expression of the target protein is increased or decreased as compared to a gRNA control that does not target Cas9 to that gene.

[0239] In some embodiments, the efficacy of editing with a particular gRNA is determined by the edits present at the target position in the genome after delivery of Cas9 and the gRNA (either sgRNA or crRNA and trRNA-containing dgRNA). In some embodiments, the efficacy of editing with a particular gRNA is measured by next-generation sequencing. In some embodiments, the percentage of editing of the target region of interest is determined. In some embodiments, the total number of sequence reads having nucleotide insertions or deletions in the target region of interest relative to the total number of sequence reads is measured after delivery of the gRNA and Cas9. In some embodiments, the present invention includes a method of increasing the efficacy of gene editing, comprising administering or delivering to a cell any one of the modified gRNAs described herein, wherein the percentage of gene editing is increased as compared to an unmodified control gRNA.

[0240] In some embodiments, the efficiency of editing using a particular gRNA is measured by the presence of nucleotide insertions or deletions introduced by the success of gene editing. In some embodiments, the present invention includes a method of creating nucleotide insertions or deletions in a gene, including administering or delivering to a cell any one of the modified gRNAs described herein, wherein the nucleotides are inserted or deleted as compared to an unmodified control gRNA. In some embodiments, the activities of Cas9 and the gRNA are tested in a biochemical assay. In some embodiments, the activities of Cas9 and the gRNA are tested in a cell-free cleavage assay. In some embodiments, the activities of Cas9 and the gRNA are tested in Neuro2A cells.

[0241] In some embodiments, Cas9 and a dgRNA comprising an sgRNA or a modified crRNA and / or trRNA exhibit similar, greater, or reduced activity as compared to a dgRNA comprising an unmodified sgRNA or an unmodified crRNA and trRNA. In some embodiments, Cas9 and a dgRNA comprising a modified sgRNA or a modified crRNA and / or trRNA exhibit enhanced activity as compared to a dgRNA comprising an unmodified sgRNA or an unmodified crRNA and trRNA.

[0242] 2. In Vivo Measurement of Cas Efficacy In some embodiments, the activity of the modified gRNA is measured after in vivo administration of an LNP comprising the modified gRNA and a Cas protein or an mRNA encoding a Cas protein.

[0243] In some embodiments, the in vivo efficacy of a gRNA or composition provided herein is determined by the editing efficacy measured in DNA extracted from a tissue (e.g., liver tissue) after administration of the gRNA and Cas9.

[0244] 3. In Vivo Measurement of Immune System Activation The modifications of gRNAs disclosed herein may reduce the immune response of a subject to in vivo administration of the gRNA. In some embodiments, activation of the subject's immune response is measured by the serum concentration of cytokine(s) after in vivo administration of a dgRNA comprising an sgRNA or trRNA and crRNA, along with Cas9 mRNA or protein (e.g., formulated in an LNP). In some embodiments, the cytokine is interferon-alpha (IFN-alpha), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and / or tumor necrosis factor-alpha (TNF-alpha). In some embodiments, the invention includes a method of reducing the immune response of a subject to delivery of a gRNA, comprising administering any one of the gRNAs disclosed herein, wherein the gRNA produces a reduced response by the subject's immune system after administration. In some embodiments, the invention includes a method of reducing activation of the subject's immune system after administration, as compared to an unmodified control gRNA.

[0245] In some embodiments, administration of a Cas RNP or Cas9 mRNA along with a modified gRNA (e.g., an sgRNA or dgRNA) produces a lower serum concentration(s) of immune cytokines as compared to administration of an unmodified sgRNA. In some embodiments, the invention includes a method of reducing the serum concentration of immune cytokines in a subject, comprising administering any one of the gRNAs disclosed herein, wherein the gRNA produces a lower concentration of immune cytokines in the subject's serum as compared to an unmodified control gRNA.

[0246] This description and the exemplary embodiments are not to be construed in a limiting sense. For the purposes of this specification and the appended claims, unless otherwise indicated, all amounts, percentages, or ratios used in this specification and the claims, and all other numerical representations, are to be understood as being modified in all instances by the term "about" if not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the reported number of significant digits and in reference to ordinary rounding techniques.

[0247] As used in this specification and the appended claims, the singular forms "a", "an", and "the" and any singular use of any word are noted to include plural referents unless expressly and unambiguously limited to one referent. As used herein, the term "comprising" and its grammatical variations are intended to be non-limiting, and the recitation of items in a list is not to be construed as excluding other like items that may be substituted or added to the listed items.

Examples

[0248] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0249] Example 1 - Materials and Methods A. Synthetic guide RNA (gRNA) The gRNA was chemically synthesized in both dual (dgRNA, i.e., crRNA and trRNA) and single-guide (sgRNA) formats using the modified nucleotides and linkages provided by the vendor in Table 4.

[0250] B. In vitro transcription (「IVT」) of Cas9 mRNA Capped and polyadenylated Cas9 mRNA containing N1-methylpseudouridine was generated by in vitro transcription using linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing the T7 promoter and a 100 nucleotide (nt) poly(A / T) region was linearized with XbaI and obtained from a commercial manufacturer. The IVT reaction for generating modified mRNA of Cas9 was incubated at 37 °C for 4 hours under the following conditions: 50 ng / μL of linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methylpseudouridine triphosphate (Trilink); 10 mM of ARCA (Trilink); 5 U / μL of T7 RNA polymerase (NEB); 1 U / μL of murine ribonuclease inhibitor (NEB); 0.004 U / μL of inorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer. After 4 hours of incubation, TURBO DNase (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 the enzymes and nucleotides using a standard protocol including a silica-binding column such as the MegaClear Transcription Clean-up kit (ThermoFisher) or a precipitation step using EtOH containing NaOAc after LiCl. The transcript concentration was determined by measuring the absorbance at 260 nm (Nanodrop), and the transcripts were analyzed by capillary electrophoresis using a Bioanalyzer (Agilent).

[0251] C. Transfection of Cas9 mRNA and gRNA in Neuro2A cells Mouse cell line Neuro2A was cultured in DMEM medium supplemented with 10% fetal bovine serum and plated at a density of 15,000 cells / well in 96-well plates 24 hours prior to 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. Both Cas9 mRNA and gRNA were mixed separately with the diluted Lipofectamine-2000 at 1:1 (v / v) to generate two lipoplexes. After a 5-minute incubation, the lipoplexes were added sequentially to the cells to a final concentration of 100 ng of Cas9 mRNA / well and 0.4 μL of total lipofection reagent. For each experiment, guides were tested at two dose levels 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, e.g., 25 nM of crRNA and 25 nM of trRNA to generate a total dual guide of 25 nM. Cells were lysed 24 hours after transfection and the lysate was used directly in a PCR reaction for analysis of editing by NGS.

[0252] Cas9 mRNA with 1xNLS (SEQ ID NO: 359):

[0253] Cas9 mRNA with 2xNLS and HA tag (SEQ ID NO: 360):

[0254] D. Primary hepatocytes Primary mouse hepatocytes (PMH) (Gibco) were cultured according to the manufacturer's protocol (Invitrogen, Protocol 11.28.2012). Briefly, the cells were thawed, resuspended in hepatocyte thawing medium (Gibco, product number CM7000) containing additives, and then centrifuged at 100 g for 10 minutes. The supernatant was discarded, and the pelleted cells were resuspended in hepatocyte plating medium (Invitrogen, product numbers A1217601 and CM3000) with the additive pack added. The cells were counted and plated at a density of 15,000 cells / well in a 96-well plate coated with Bio-coat collagen I (ThermoFisher, product number 877272) and incubated at 37 °C and 5% CO2 atmosphere for 5 hours to form a monolayer. After 5 hours, the plating medium was removed and replaced with supplemented hepatocyte culture medium (Invitrogen, product numbers A1217601 and CM4000) containing 3% mouse serum in addition to Cas9 mRNA and guide RNA. The LNP was diluted from an initial dose level of 100 ng of Cas9 mRNA and approximately 30 nM of guide RNA per well, and serial dilutions were performed down to 0.1 ng of mRNA and 0.03 nM of guide per well. After the cells were incubated at 37 °C and 5% CO2 atmosphere for approximately 48 hours, cell lysis and NGS analysis described herein were performed.

[0255] E. Formulation of lipid nanoparticles (“LNP”) LNPs were formulated at a molar ratio (N:P) of cationic lipid amine to RNA phosphate of approximately 4.5. The lipid nanoparticle components were dissolved in 100% ethanol at the following molar ratios: 45 mol% (12.7 mM) of a cationic lipid (e.g., 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), also known as (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate; 44 mol% (12.4 mM) of a helper lipid (e.g., cholesterol); 9 mol% (2.53 mM) of a neutral lipid (e.g., DSPC); and 2 mol% (0.563 mM) of PEG (e.g., PEG2k-DMG). RNA cargo was prepared in 25 mM sodium acetate buffer at pH 4.5 at a concentration of approximately 0.45 mg / mL of RNA cargo.

[0256] The Precision Nanosystems NanoAssemblr™ Benchtop Instrument was used to form LNPs by microfluidic mixing of the lipid and RNA solutions according to the manufacturer's protocol. A 2:1 aqueous solvent to organic solvent ratio was maintained during mixing using a flow rate differential.

[0257] LNP formulation procedure A: After mixing, the LNPs were recovered, diluted in phosphate buffered saline (PBS, approximately 1:1), and then the remaining buffer was exchanged with PBS (100-fold excess of the sample volume) overnight at 4°C with gentle stirring using a 10 kDa Slide-a-Lyzer™ G2 Dialysis Cassette (ThermoFisher Scientific). The LNPs were concentrated (centrifugation at 4000 g at 4°C) using a 10 kDa Amicon spin filter 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.

[0258] LNP formulation procedure B: After mixing, the LNPs were recovered and diluted in 50 mM Tris at pH 7.5 (about 1:1), and then the LNPs were exchanged into 50 mM Tris at pH 7.5 (100-fold excess of the sample volume) at 4 °C overnight with gentle stirring using a 10 kDa Slide-a-LyzerTM G2 Dialysis Cassette (ThermoFisher Scientific). The LNPs were concentrated (centrifuged at 4 °C and 4000 g) using a 10 kDa Amicon spin filter to achieve twice the desired concentration. These concentrated LNPs were mixed 1:1 with 50 mM Tris, 90 mM NaCl, and 10% sucrose (2X TSS) at pH 7.5. Next, the resulting mixture was filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at -80 °C.

[0259] LNP formulation procedure C: RNA cargo was prepared at pH 5 in 25 mM sodium citrate, 100 mM sodium chloride at an RNA cargo concentration of approximately 0.45 mg / mL. After mixing, the LNPs were recovered in water at a ratio of 3:1. The LNPs were incubated at room temperature for 1 hour and mixed 1:1 with water. Then they were buffer-exchanged into 1X TSS (50 mM Tris, 45 mM NaCl, 5% sucrose, pH 7.5) on a PD-10 column (GE Healthcare) using the manufacturer's protocol. The LNPs were concentrated (centrifuged at 4 °C and 4000 g) using a 10 kDa Amicon spin filter to achieve the desired concentration. Next, the resulting mixture was filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at -80 °C.

[0260] F. Next-generation sequencing (“NGS”) and analysis of on-target cleavage efficiency To quantitatively determine the efficiency of editing at target positions in the genome, deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing.

[0261] PCR primers were designed around the target sites (e.g., TTR, FVII) to amplify the target genomic segments. The primer sequences are provided in Table 5 below.

Table 5

[0262] The editing percentage (e.g., "editing efficiency" or "editing percent") is defined as the total number of sequence reads with insertions or deletions relative to the total number of sequence reads including wild-type.

[0263] G. LNP Delivery in vivo CD-1 female mice in the range of 6 - 10 weeks of age were used in each study. The animals were weighed and grouped according to their body weight to prepare the dosing solution based on the average weight of the group. LNP was administered via the lateral tail vein at a volume of 0.2 mL per animal (about 10 mL per kilogram of body weight). The animals were observed for adverse effects about 6 hours after dosing. Body weight was measured 24 hours after dosing, and the animals were euthanized at various time points by exsanguination via cardiac puncture under isoflourane anesthesia. Blood was collected into serum separator tubes or tubes containing buffered sodium citrate for plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the mid-lobe of each animal for DNA extraction and analysis.

[0264] H. Cytokine Induction Analysis For this analysis, approximately 50 - 100 μL of blood was collected for serum cytokine measurement by making an incision in the tail vein. The blood was allowed to clot at room temperature for about 2 hours and then centrifuged at 1000×g for 10 minutes, after which the serum was collected. A Luminex-based magnetic bead multiplex assay (Affymetrix ProcartaPlus, product number Exp040 - 00000 - 801) for measuring IL-6, TNF-alpha, IFN-alpha, and MCP-1 was used for cytokine analysis in the collected samples. The kit reagents and standards were prepared as instructed by the manufacturer's protocol. 25 μL of mouse serum was added to wells containing 25 μL of magnetic beads coated with diluted antibody. The plate was incubated at room temperature for 2 hours and then washed. Diluted biotin antibody (50 μL) was added to the beads and incubated at room temperature for 1 hour. After washing the beads again, 50 μL of diluted streptavidin-PE was added to each well and incubated for 30 minutes. After washing the beads once more, they were suspended in 100 μL of wash buffer and read on a Bio-Plex 200 instrument (Bio-Rad). Data were analyzed using the Bioplex Manager ver.6.1 analysis package with cytokine concentrations calculated from the standard curve using a 5-parameter logistic curve fit.

[0265] I. Isolation of Genomic DNA For in vivo studies, a bead-based extraction kit, MagMAX-96 DNA Multi-Sample Kit (ThermoFisher, product number 4413020), was used according to the manufacturer's protocol, which included homogenizing the tissue in lysis buffer (approximately 400 μL per 10 mg of tissue), to extract genomic DNA from 10 mg of tissue. As described herein, all DNA samples were normalized to a concentration of 100 ng / μL for PCR and subsequent NGS analysis.

[0266] J. ELISA Analysis of Transthyretin (TTR) Blood was collected and serum was isolated as indicated. Total TTR serum levels were determined using a Mouse Prealbumin (Transthyretin) ELISA Kit (Aviva Systems Biology, product number OKIA00111). The kit reagents and standards were prepared according to the manufacturer's protocol. Mouse serum was diluted using 1× assay diluent to a final dilution of 10,000-fold. This was done by performing two sequential 50-fold dilutions, resulting in a 2500-fold dilution. A final 4-fold dilution step was performed for the 10,000-fold total sample dilution. Both standard curve dilutions (100 μL each) and the diluted serum samples were added to each well of an ELISA plate pre-coated with the capture antibody. The plate was incubated at room temperature for 30 minutes and then washed. The enzyme-antibody conjugate (100 μL per well) was added for a 20-minute incubation. Unbound antibody conjugate was removed and the plate was washed again, after which the chromogenic substrate solution was added. The plate was incubated for 10 minutes and then 100 μL of stop solution, for example, sulfuric acid (approximately 0.3 M), was added. The plate was read on a SpectraMax M5 plate reader at an absorbance of 450 nm. Serum TTR levels were calculated from the standard curve using SoftMax Pro software ver. 6.4.2 with a 4-parameter logistic curve fit. The final serum values were adjusted for the assay dilution.

[0267] Example 2 - Manipulation and in vitro testing of modified gRNAs As shown in Table 4, modified gRNAs were designed in a dual guide format (dgRNA). As a result, both modified crRNA and trRNA were designed and chemically synthesized to pair the modified and unmodified components that form the dgRNA. These pairings were transfected into Neuro2A cells at the concentrations indicated in the figure, and the editing efficiency (e.g., percent editing) was measured by NGS as described in Example 1.

[0268] Certain modified crRNAs in Table 4 targeting the mouse TTR gene were transfected with Cas9 mRNA and unmodified trRNA (TR000002). The guides tested included SEQ ID NOs: 1-18. As shown in Figure 1, some of the modified crRNAs (along with the unmodified trRNA) conferred similar or enhanced activity compared to the unmodified control, while other modified crRNAs decreased activity.

[0269] In parallel, the modified trRNAs in Table 4 were transfected with Cas9 mRNA together with an unmodified crRNA (CR000686) targeting the same sequence of the mouse TTR gene. The guides tested included SEQ ID NOs: 188-200, and 204. As shown in Figure 2, many of the modified trRNAs (along with the unmodified crRNA) conferred similar or enhanced activity compared to the unmodified control, while some of the modified trRNAs decreased activity.

[0270] In addition to substitutions of chemically modified nucleotides, some of the pairing of the crRNAs and trRNAs tested were also engineered by sequence substitution, resulting in, for example, G-C base pair formation not seen in the parental sequences. The guides tested included SEQ ID NOs: 15 and 201; 16 and 202; 1 and 188. As shown in Figure 3, one such pairing (SEQ ID NOs: 16 and 202) resulted in similar or enhanced activity compared to the unmodified control, while two of the pairings decreased activity.

[0271] Next, the pairing of the modified crRNAs and modified trRNAs in Table 4 was tested. As shown in Figure 4, some of the pairing of the modified crRNAs with the modified trRNAs conferred similar or enhanced activity compared to the unmodified control, while some of the pairing decreased activity. In Figure 4, the column headings represent the different trRNAs used in the experiment, and the row headings represent the different crRNAs used. To determine the combinations used in the experiment, match the columns to the rows. TR000002 and CR000686 are the unmodified controls (see the bottom right column).

[0272] Based on the design of dgRNA, as depicted in Table 4 and Figure 15D, corresponding single-guide RNAs (sgRNAs) were engineered featuring certain aspects of modified crRNAs and trRNAs. These sgRNAs of SEQ ID NOs: 228 - 234 were also tested in Neuro2A cells, and as shown in Figure 5, each of the modified sgRNAs exhibited activity equivalent to that of a control (G0000209; SEQ ID NO: 228) containing only 5’ and 3’ end modifications.

[0273] A similar set of experiments was performed for the additional dgRNA guides depicted in Table 4 and Figure 6. The guides tested included SEQ ID NOs: 32 - 47, and 1. These also involved transfecting modified crRNAs targeting the mouse TTR gene along with Cas9 mRNA and unmodified trRNA (TR000002). As shown in Figure 6, some of the modified crRNAs, along with unmodified trRNA, conferred similar or enhanced activity compared to an unmodified control (CR000686), while other modified crRNAs decreased activity.

[0274] In parallel, as shown in Figure 7, the modified trRNAs in Table 4 were transfected with Cas9 mRNA together with an unmodified crRNA (CR000686) targeting the same sequence of the mouse TTR gene. The guides tested included SEQ ID NOs: 205 - 222, and 1. As shown in Figure 7, many of the modified trRNAs, along with the unmodified crRNA, conferred similar or enhanced activity compared to an unmodified control (TR000002), while some of the modified trRNAs decreased activity.

[0275] In addition to the substitution of chemically modified nucleotides, some of the pairings of the tested crRNAs and trRNAs from Table 4 were also manipulated by sequence substitution, resulting in, for example, G-C base pair formation or G-U mismatches ("GU wobbles") not seen in the parental sequences. As shown in Figure 8, some of the modifications and pairings conferred similar or enhanced activity compared to the unmodified controls, while some (e.g., "GU wobbles" or mismatched pairings) decreased activity. Figure 8 shows the results using the crRNA guides shown in SEQ ID NOs: 48-52, and 1, and the trRNA guides shown in SEQ ID NOs: 223-227 and 188.

[0276] Next, selected pairings of the modified crRNAs and modified trRNAs from Table 4 were tested as shown in Figure 9. Some of the pairings of the modified crRNAs with the modified trRNAs conferred similar or enhanced activity compared to the unmodified controls, while some of the pairings decreased activity. In Figure 9, the column headings represent the different trRNAs used in the experiment, and the row headings represent the different crRNAs used. To determine the combinations used in the experiment, match the columns to the rows. The unmodified controls are TR000002, and CR000686.

[0277] Some of the modified gRNAs (dgRNAs and sgRNAs) from Table 4 were also tested in purely biochemical assays (i.e., cell-free cleavage assays). Interestingly, many of the modified gRNAs that were generally inactive in Neuro2A cells were active in the biochemical assays, indicating that such biochemical assays may not predict the activity of modified gRNAs in cells (data not shown).

[0278] Example 3. Further Testing of Modified gRNAs Against Other Targets Since it was established that certain modifications affect the activity of gRNAs, we next tested whether these modifications would affect activity when (1) targeting separate sequences within the same gene or (2) targeting sequences in different genes. As a result, gRNAs targeting not only a different sequence in the mouse TTR gene but also a sequence in the mouse factor VII (FVII) gene were engineered and synthesized to have a particular modification pattern tested in Example 2 (see Table 4). These gRNAs were transfected into Neuro2A cells at the concentrations indicated in the figure, and the editing efficiency (e.g., percent editing) was measured by NGS as described in Example 1.

[0279] The modified crRNAs in Table 4 that target either the mouse TTR gene (a different sequence than that targeted in Example 2) or the mouse FVII gene were transfected with Cas9 mRNA and unmodified trRNA (TR000002). The guides tested included those shown in FIGS. 12A and 12B. Some of the modified crRNAs (along with the unmodified trRNA) conferred similar or enhanced activity compared to the unmodified control, while other modified crRNAs decreased activity.

[0280] In parallel, the modified trRNAs from Table 4 were transfected with Cas9 mRNA together with an unmodified crRNA targeting the same sequence of the mouse TTR gene (CR000705; a different sequence than that targeted in Example 2) or the same sequence of the mouse FVII gene (CR000657). As shown in FIGS. 13A and 13B, many of the modified trRNAs (along with the unmodified crRNA) conferred similar or enhanced activity compared to the unmodified control, while some of the modified trRNAs decreased activity. This data indicates that certain modification patterns tended to have similar effects across different sequences.

[0281] Based on the design of the dgRNA described above, the corresponding single-guide RNA (sgRNA) was engineered, featuring certain modifications to the crRNA and trRNA. See Table 4. These sgRNAs were also tested in Neuro2A cells. The results are shown in Figures 10 (mouse TTR) and 11 (mouse FVII). These experiments show that some modification patterns result in similar effects even when targeting different genes.

[0282] Example 4. Testing of Modified gRNA in vivo After in vitro testing, modified sgRNAs were delivered to animals in six separate studies to determine whether the modifications confer any benefit for in vivo editing.

[0283] As described in Example 1, LNPs were formulated containing IVT Cas9 mRNA together with chemically modified sgRNAs (targeting TTR or FVII). The mRNA:sgRNA ratio was approximately 1:1 on a mass basis of the RNA components. Unless otherwise indicated, the Cas9 mRNA used in the studies described in this example had the sequence of SEQ ID NO: 360, and the LNPs were formulated using the above-described LNP formulation procedure A.

[0284] In one experiment, single-dose LNPs at 2 mg / kg were administered to mice (n = 5 per group), and blood was collected 4 hours after administration for serum cytokine analysis. At dissection 7 days after administration, liver and blood were collected for NGS measurement of editing efficiency and analysis of serum TTR, 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 performed on each (see Figures 14A - D and 15A - E; Table 4, SEQ ID NOs: 228 - 234). G000209 (two lots were tested) functioned as a less modified control and had only 2'-O-methyl modifications and phosphorothioate linkages at each of the three terminal nucleotides at both the 5' and 3' ends of the sgRNA and between them (see Figure 15D).

[0285] The results shown in FIGS. 14A - D indicate that more heavily modified sgRNAs tended to induce lower responses for each cytokine assayed compared to the less modified G000209 control. The more heavily modified sgRNAs also had editing percentages reaching about 60% for two of the more heavily modified sgRNAs (e.g., G000263 and G000267), compared to about 44 - 47% for the less modified control (G000209 lot), conferring a higher editing efficiency in the liver of treated animals (FIG. 15A). Importantly, the knockdown of serum TTR levels was equal to or significantly higher than that of the less modified control, so the editing efficiency correlated with the phenotypic change (e.g., see G000263 and G000267 versus the G000209 lot in FIGS. 15A - 15B). The differences between the terminal modification G000209 and the highly modified G000267 are summarized in FIGS. 15D and 15E (2'-O-Me modified nucleotides are shown in bold and * represents phosphorothioate linkages).

[0286] In another in vivo study, three sgRNAs targeting separate sequences in the mouse TTR gene were tested. Mice (n = 5 per group) were administered single doses of LNP at 2 mg / kg, 1 mg / kg, or 0.3 mg / kg. Blood was collected 4 hours after administration for serum cytokine analysis. At dissection 7 days after administration, liver and blood were collected for NGS measurement of editing efficiency and analysis of serum TTR, respectively. In this study, each of the sgRNAs, one sgRNA was completely unmodified (G000201 (SEQ ID NO: 243)), another sgRNA had only terminal modifications with 2'-O-methyl modifications and phosphorothioate linkages at three terminal nucleotides at each of the 5' and 3' ends of the sgRNA, and between them (G000211 (SEQ ID NO: 241)), and the third sgRNA had the same modification pattern as G000267 in a previous in vivo study (G000282 (SEQ ID NO: 242)) and targeted the same sequence in the TTR gene (a different sequence from that targeted in the previous in vivo study).

[0287] As shown in FIGS. 16A - 16D, each of the sgRNAs resulted in similar responses in a dose - dependent manner for each of the cytokines tested. For editing efficiency, the unmodified sgRNA (G000201 (SEQ ID NO: 243)) conferred little editing in vivo, while the heavily modified sgRNA (G000282 (SEQ ID NO: 242)) conferred levels reaching approximately 60% at a dose of 2 mg / kg, which was significantly higher than the levels achieved with the less modified sgRNA (G000211 (SEQ ID NO: 241)) (FIGS. 17A and B). Similar to the previous in vivo study, the level of editing correlated with the amount of serum TTR knockdown (FIGS. 17C and D).

[0288] Using another set of three sgRNAs that target different TTR sequences in the mouse TTR gene (targeting sequences different from those targeted in two previous in vivo studies), a study similar to the second in vivo study was then performed. Mice (n = 5 per group) were administered single doses of LNP at 2 mg / kg, 1 mg / kg, or 0.3 mg / kg. Blood was collected 4 hours after administration for serum cytokine analysis. At dissection 7 days after administration, liver and blood were collected for NGS measurement of editing efficiency and analysis of serum TTR, respectively. In this study, each of the sgRNAs, one sgRNA was completely unmodified (G000285; (SEQ ID NO: 332)), another sgRNA had only terminal modifications with 2'-O-methyl modifications and phosphorothioate linkages at each of three terminal nucleotides at both the 5' and 3' ends of the sgRNA, and between them (G000269 (SEQ ID NO: 330)), and the third sgRNA had the same modification pattern as G000267 and G000282 in the two previous in vivo studies (G000283 (SEQ ID NO: 331)) and targeted the same sequence in the TTR gene (a sequence different from that targeted in the two previous in vivo studies).

[0289] In this study, the unmodified sgRNA (G000285 (SEQ ID NO: 332)) conferred little editing in vivo, whereas the highly modified sgRNA (G000283 (SEQ ID NO: 331)) conferred levels reaching approximately 60% at a dose of 2 mg / kg, which was significantly higher than the levels achieved with the less modified sgRNA (G000269 (SEQ ID NO: 330)) (FIGS. 18A - 18B). Similar to the previous in vivo studies, the level of editing correlated with the amount of serum TTR knockdown (FIG. 18C).

[0290] In the 4th in vivo study, the effect of the modification to the gRNA was evaluated for another gene (FVII). For the within-study comparison, two of the sgRNAs tested in the 1st in vivo study were included (G000209 and G000267). Mice (n = 5 per group) were administered a single dose of LNP at 2 mg / kg, 1 mg / kg, or 0.3 mg / kg, and blood was collected 4 hours after administration for serum cytokine analysis. At necropsy 6 days after administration, the liver was collected for NGS measurement of the editing efficiency. In this study, each of the sgRNAs targeted the same sequence in the TTR or FVII gene, and for each, one sgRNA had only terminal modifications with both 2'-O-methyl modification and phosphorothioate linkages at each of three terminal nucleotides at both the 5' and 3' ends of the sgRNA, and between them (G000208 (SEQ ID NO: 286) for FVII, G000209 for TTR), and the second sgRNA had the same modification pattern as G000267, G000282, and G000283 in the previous in vivo study (G000373 (SEQ ID NO: 287) for FVII; G000267 (SEQ ID NO: 234) for TTR).

[0291] As shown in FIGS. 19A-19D, each of the sgRNAs resulted in a similar response in a dose-dependent manner for each of the cytokines tested. For the editing efficiency, the more heavily modified sgRNA targeting FVII (G000373 (SEQ ID NO: 287)) had an increase in editing efficiency compared to the less modified version (G000208 (SEQ ID NO: 286)) across each of the doses tested (FIG. 18A). These results were also observed for the sgRNAs targeting TTR (FIGS. 20A-20B).

[0292] In another in vivo study, 10 additional sgRNAs targeting sequences in the same mouse TTR gene as G000282 were tested. G000282 was also included in the study for comparison purposes. Mice (n = 5 per group) were administered a single dose of 1 mg / kg or 0.5 mg / kg of LNP. The LNP used in this study was formulated using LNP formulation procedure B described above. At dissection 7 days after administration, liver and blood were collected for NGS measurement of editing efficiency and analysis of serum TTR, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene. The modification patterns of each of the sgRNAs tested were diverse and included 2'-OMe, 2'-F, and PS modifications in the 5' end, 3' end, hairpin 1, hairpin 2, nexus, lower stem, bulge, and upper stem of the sgRNA. The results of this study, including the editing % (Figure 22A), mean and standard deviation of editing (Figure 22B), and serum TTR levels (Figure 22C), are shown in Figures 22A - 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, including the editing % (Figure 24A), dose - response curve (Figure 24B), and EC50 value (Figure 24C), are shown in Figures 24A - 24C.

[0293] In another in vivo study, 13 additional sgRNAs targeting the same sequence as G000282 in the mouse TTR gene were tested. G000282 was also included in the study for comparison purposes. Mice (n = 5 per group) were administered a single dose of 1 mg / kg of LNP. The LNP used in this study was formulated using the above-described LNP formulation procedure C. The Cas9 mRNA used in this study had the sequence of SEQ ID NO: 359. Blood was collected 4 hours after administration for serum cytokine analysis. At dissection 7 days after administration, liver and blood were collected for NGS measurement of editing efficiency and analysis of serum TTR, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene. The sgRNAs tested contained additional 2'-OMe and PS modifications in the 5' end, 3' end, hairpin 1, hairpin 2, and upper stem of the sgRNA. The results of this study, including the editing percentage (Figure 23A), average percentage of editing (Figure 23B), and serum TTR levels (Figure 23C), are shown in Figures 23A - 23C. May include the following aspects. [1] A single guide RNA (sgRNA) comprising a 5’ end modification and a. One or more modifications in one or more of the upper stem region, b. Hairpin 1 region, and c. Hairpin 2 region, wherein the 5’ end modification comprises at least two phosphorothioate (PS) linkages in the first 7 nucleotides at the 5’ end of the 5’ terminus, sgRNA. [2] The sgRNA according to [1] above, wherein at least one modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide. [3] The sgRNA according to [1] or [2] above, wherein at least one modification comprises a 2’-fluoro (2’-F) modified nucleotide. [4] The sgRNA according to any one of [1] to [3] above, wherein at least one modification comprises a phosphorothioate (PS) bond between nucleotides. [5] The sgRNA according to any one of [1] to [4] above, comprising one or more modifications in the upper stem region. [6] The sgRNA according to [5] above, comprising a modification in US1-US12. [7] The sgRNA according to any one of [1] to [6] above, comprising one or more modifications in the hairpin 1 region. [8] The sgRNA according to [7] above, comprising a modification in H1-1. [9] The sgRNA according to any one of [1] to [8] above, comprising one or more modifications in the hairpin 2 region.

[10] The sgRNA according to [9] above, which contains a modification in H2-1.

[11] The sgRNA according to any one of [1] to

[10] above, which contains a modification in H1-1 to H1-12.

[12] The sgRNA according to any one of [1] to

[11] above, which contains a modification in H2-1 to H2-15.

[13] The sgRNA according to any one of [1] to

[12] above, which contains one or more modifications in each of the upper stem region, the hairpin 1 region, and the hairpin 2 region.

[14] The sgRNA according to any one of [1] to

[13] above, which contains a modified nucleotide between the hairpin 1 region and the hairpin 2 region.

[15] The sgRNA according to any one of [1] to

[14] above, which further contains a lower stem region with a modification.

[16] The sgRNA according to any one of [1] to

[15] above, which further contains a 3'-terminal region with a modification.

[17] The sgRNA according to

[16] above, which further contains a 3'-terminal modification in the 3'-terminal.

[18] The sgRNA according to

[17] above, wherein at least two of the last four nucleotides at the 3'-terminal of the 3'-terminal are modified.

[19] The sgRNA according to

[17] above, wherein at least two of the last four nucleotides at the 3'-terminal of the 3'-terminal are modified with 2'-O-Me, 2'-F, or 2'-O-moe.

[20] The sgRNA according to any one of

[17] to

[19] above, which further contains a phosphorothioate (PS) bond between one or more of the last four nucleotides at the 3'-terminal of the 3'-terminal.

[21] The sgRNA according to any one of [1] to

[20] above, which further contains a bulge region with a modification.

[22] The sgRNA according to any one of [1] to

[21] above, which further contains a nexus region with a modification.

[23] The sgRNA according to any one of [1] to

[22] above, wherein at least the first three nucleotides at the 5'-terminal of the 5'-terminal and the last three nucleotides at the 3'-terminal of the 3'-terminal are modified.

[24] The sgRNA according to any one of [1] to

[23] above, wherein the first 4 nucleotides at the 5'-end of the 5'-terminus and the last 4 nucleotides at the 3'-end of the 3'-terminus are linked by phosphorothioate (PS) bonds.

[25] The sgRNA according to

[24] above, wherein the terminal modification contains 2'-O-Me.

[26] The sgRNA according to

[24] above, wherein the terminal modification contains 2'-F.

[27] The sgRNA according to any one of [1] to

[26] above, wherein the first 4 nucleotides at the 5'-end of the 5'-terminus and the last 4 nucleotides at the 3'-end of the 3'-terminus are linked by PS bonds, and the first 3 nucleotides at the 5'-end of the 5'-terminus and the last 3 nucleotides at the 3'-end of the 3'-terminus contain 2'-O-Me modifications.

[28] The sgRNA according to any one of [1] to

[26] above, wherein the first 4 nucleotides at the 5'-terminus and the last 4 nucleotides at the 3'-terminus are linked by PS bonds, and the first 3 nucleotides at the 5'-terminus and the last 3 nucleotides at the 3'-terminus contain 2'-O-Me, 2'-F, and / or 2'-O-moe modifications.

[29] The sgRNA according to any one of [1] to

[28] above, wherein LS1, LS6, LS7, LS8, LS11, and / or LS12 are modified with 2'-O-Me.

[30] The sgRNA according to any one of [1] to

[29] above, wherein each of the nucleotides in the bulge region is modified with 2'-O-Me.

[31] The sgRNA according to any one of [1] to

[29] above, wherein at least 50% of the nucleotides in the bulge region are modified with 2'-O-Me.

[32] The sgRNA according to any one of [1] to

[31] above, wherein each of the nucleotides in the upper stem region is modified with 2'-O-Me.

[33] The sgRNA according to any one of [1] to

[32] above, wherein N16, N17, and / or N18 in the nexus region are modified with 2'-O-Me.

[34] The sgRNA according to any one of [1] to

[32] above, wherein N15, N16, N17, and / or N18 in the nexus region are modified.

[35] The sgRNA according to

[33] or

[34] above, wherein the modification in the nexus region is selected from 2'-O-Me and 2'F.

[36] The sgRNA according to any one of

[32] to

[35] above, wherein N16, N17, and N18 are linked by PS bonds.

[37] The sgRNA according to any one of [1] to

[36] above, wherein each of the nucleotides in the hairpin 1 region is modified with 2'-O-Me.

[38] The sgRNA according to any one of [1] to

[37] above, wherein each of the nucleotides in the hairpin 2 region is modified with 2'-O-Me.

[39] A single guide RNA (sgRNA) having 2'-O-Me modified nucleotides at the following positions: a. The first 3 nucleotides at the 5' end of the 5' terminus; b. LS1, LS6, LS7, LS8, LS11, and / or LS12 in the lower stem region; c. B1 and / or B2 in the bulge region; d. Each nucleotide in the upper stem region; e. N16, N17, and / or N18 in the nexus region; f. Each nucleotide in the hairpin 1 region; g. Each nucleotide in the hairpin 2 region; h. The last 4 nucleotides at the 3' end.

[40] The sgRNA according to

[39] above, wherein B3 - B6 are modified with 2'-O-Me.

[41] The sgRNA according to

[39] above, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' end of the 3' terminus.

[42] The sgRNA according to any one of [1] to

[41] above, wherein LS9 and LS10 are modified with, for example, 2'-F or 2'-OMe.

[43] The sgRNA according to any one of [1] to

[42] above, wherein N15, N16, N17, and N18 are modified with, for example, 2'-F or 2'-OMe.

[44] The sgRNA according to any one of [1] to

[43] above, wherein H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 are modified with 2'-F.

[45] The sgRNA according to any one of [1] to

[44] above, wherein the second last, third last, and fourth last nucleotides at the 3' terminus are modified with 2'-F.

[46] A single guide RNA (sgRNA) comprising 2'-F modified nucleotides at the following positions: a. LS9 and LS10 in the lower stem region, and b. N15, N16, N17, and N18 in the nexus region, and c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 in the hairpin 2 region.

[47] The sgRNA according to

[46] above, further comprising 2'-F modified nucleotides at the second last, third last, and fourth last nucleotides at the 3' terminus.

[48] The sgRNA according to

[46] or

[47] above, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' terminus of the 5' end and three PS bonds connecting the last 4 nucleotides at the 3' terminus of the 3' end.

[49] The sgRNA according to any one of

[46] to

[48] above, further comprising 2'-O-Me or 2'-F modified nucleotides at the first 3 nucleotides at the 5' terminus of the 5' end and 2'-O-Me or 2'-F modified nucleotides at three of the last 4 nucleotides at the 3' terminus of the 3' end.

[50] A single guide RNA (sgRNA) comprising: a. 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' terminus of the 5' end, and b. 2'-O-Me modified nucleotides in US1 to US12, and c. 2'-O-Me modified nucleotides in H1-1 to H1-12, and d. 2'-O-Me modified nucleotides in H2-1 to H2-15, and e. 2'-O-Me modified nucleotides at the last 4 nucleotides at the 3' terminus of the 3' end.

[51] The sgRNA according to

[50] above, further comprising a 2'-O-Me modified nucleotide in LS1 and / or LS6.

[52] The sgRNA according to

[50] or

[51] above, further comprising a 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2.

[53] A single guide RNA (sgRNA) comprising: a. a 2'-O-Me modified nucleotide in the first 3 nucleotides at the 5'-end of the 5'-terminus; b. 2'-F modified nucleotides in LS1-LS6; c. 2'-O-Me modified nucleotides in US1-US12; d. 2'-O-Me modified nucleotides in H1-1-H1-12; e. a 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2; f. 2'-O-Me modified nucleotides in H2-1-H2-15; g. a 2'-O-Me modified nucleotide in the last 4 nucleotides at the 3'-end of the 3'-terminus.

[54] A single guide RNA (sgRNA) comprising: a. a 2'-O-Me modified nucleotide in the first 3 nucleotides at the 5'-end of the 5'-terminus; b. 2'-F modified nucleotides in LS2-LS5; c. 2'-O-Me modified nucleotides in LS1 and LS6; d. 2'-O-Me modified nucleotides in US1-US12; e. 2'-O-Me modified nucleotides in H1-1-H1-12; f. a 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2; g. 2'-O-Me modified nucleotides in H2-1-H2-15; h. a 2'-O-Me modified nucleotide in the last 4 nucleotides at the 3'-end of the 3'-terminus.

[55] A single guide RNA (sgRNA) comprising: a. a 2'-O-Me modified nucleotide in the first 3 nucleotides at the 5'-end of the 5'-terminus; b. 2'-O-Me modified nucleotides in US1-US12; c. 2'-O-Me modified nucleotides in LS7, LS8, LS11, and LS12; d. 2'-O-Me modified nucleotides in H1-1-H1-12; e. a 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2; f. 2'-O-Me modified nucleotides in H2-1-H2-15; An sgRNA comprising a 2'-O-Me modified nucleotide at the last 4 nucleotides at the 3'-end of the 3'-terminus.

[56] A single guide RNA (sgRNA) comprising: a. A 2'-O-Me modified nucleotide at the first 3 nucleotides at the 5'-end of the 5'-terminus; b. 2'-O-Me modified nucleotides in US1 to US12; c. 2'-O-Me modified nucleotides in LS7, LS8, LS11, and LS12; d. 2'-F modified nucleotides in LS9 and LS10; e. 2'-O-Me modified nucleotides in H1-1 to H1-12; f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; g. 2'-O-Me modified nucleotides in H2-1 to H2-15; h. A 2'-O-Me modified nucleotide at the last 4 nucleotides at the 3'-end of the 3'-terminus.

[57] A single guide RNA (sgRNA) comprising: a. A 2'-O-Me modified nucleotide at the first 3 nucleotides at the 5'-end of the 5'-terminus; b. 2'-O-Me modified nucleotides in US1 to US12; c. 2'-O-Me modified nucleotides in LS8, LS10, and LS12; d. 2'-O-F modified nucleotides in LS7, LS9, and LS11; e. 2'-O-Me modified nucleotides in H1-1 to H1-12; f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; g. 2'-O-Me modified nucleotides in H2-1 to H2-15; h. A 2'-O-Me modified nucleotide at the last 4 nucleotides at the 3'-end of the 3'-terminus.

[58] A single guide RNA (sgRNA) comprising: a. A 2'-O-Me modified nucleotide at the first 3 nucleotides at the 5'-end of the 5'-terminus; b. 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12; c. 2'-O-Me modified nucleotides in US1 to US12; d. 2'-O-Me modified nucleotides in H1-1 to H1-12; e. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; f. 2'-O-Me modified nucleotides in H2-1 to H2-15; g. An sgRNA comprising a 2'-O-Me modified nucleotide at the last 4 nucleotides at the 3' end of the 3' terminus.

[59] A single guide RNA (sgRNA) comprising: a. A 2'-O-Me modified nucleotide at the first 3 nucleotides at the 5' end of the 5' terminus; b. 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12; c. 2'-F modified nucleotides in LS9 and LS10; d. 2'-O-Me modified nucleotides in US1-US12; e. 2'-O-Me modified nucleotides in H1-1-H1-12; f. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; g. 2'-O-Me modified nucleotides in H2-1-H2-15; h. A 2'-O-Me modified nucleotide at the last 4 nucleotides at the 3' end of the 3' terminus.

[60] A single guide RNA (sgRNA) comprising: a. A 2'-O-Me modified nucleotide at the first 3 nucleotides at the 5' end of the 5' terminus; b. 2'-O-Me modified nucleotides in US1-US12; c. 2'-O-Me modified nucleotides in H1-1-H1-12; d. 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2; e. 2'-O-Me modified nucleotides in H2-1-H2-8; f. 2'-F modified nucleotides in H2-9-H2-15; g. 2'-F modified nucleotides at the second last, third last, and fourth last nucleotides at the 3' terminus; h. A 2'-O-Me modified nucleotide at the last nucleotide at the 3' end of the 3' terminus.

[61] A single guide RNA (sgRNA) comprising: a. A 2'-O-Me modified nucleotide at the first 3 nucleotides at the 5' end of the 5' terminus; b. 2'-O-Me modified nucleotides in US1-US12; c. 2'-O-Me modified nucleotides in H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12; d. 2'-F modified nucleotides in H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11; e. The 2'-F modified nucleotides between hairpin 1 and hairpin 2, and f. The 2'-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10, H2-12; and H2-14; and g. The 2'-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9, H2-11; H2-13, and H2-15; and h. The 2'-F modified nucleotides at the second last and fourth last nucleotides at the 3' terminus, and i. The 2'-O-Me modified nucleotides at the third last and last nucleotides at the 3' terminus of the 3' end, comprising the sgRNA.

[62] The sgRNA according to any one of

[50] to

[61] above, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' terminus of the 5' end and three PS bonds connecting the last 4 nucleotides at the 3' terminus of the 3' end.

[63] The sgRNA according to any one of

[50] to

[61] above, further comprising at least one phosphorothioate (PS) bond within the first 7 nucleotides at the 5' terminus of the 5' end.

[64] A single guide RNA (sgRNA), a. The 2'-O-Me modified 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 b. The 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, comprising the sgRNA.

[65] The sgRNA according to

[64] above, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' terminus of the 5' end and three PS bonds connecting the last 4 nucleotides at the 3' terminus of the 3' end.

[66] a. The 2'-O-Me modified nucleotides at the last and third last nucleotides at the 3' terminus of the 3' end, and b. The sgRNA according to the above

[64] or

[65] , further comprising a 2'-F modified nucleotide at the second last and fourth last nucleotides at the 3' end of the 3' terminal.

[67] An sgRNA comprising any one of SEQ ID NOs: 228 to 332, including the modifications in Table 4.

[68] An sgRNA comprising any one of SEQ ID NOs: 235 to 240, 265 to 285, and 309 to 329, including the modifications in Table 4.

[69] An sgRNA comprising SEQ ID NO: 240.

[70] An sgRNA comprising SEQ ID NO: 240, including the modifications in Table 4.

[71] An sgRNA comprising SEQ ID NO: 242, including the modifications in Table 4.

[72] An sgRNA comprising the modifications of SEQ ID NO: 242, as depicted in Table 4.

[73] An sgRNA comprising SEQ ID NO: 358.

[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 any one nucleic acid 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 as or equivalent to the modification shown in the reference sequence identifier in Table 4.

[75] The sgRNA according to any one of the above

[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.

[75] The sgRNA according to any one of the above [1] to

[74] , further comprising at least three PS bonds connecting the nucleotides in the hairpin 1 region.

[76] The sgRNA according to any one of the above [1] to

[75] , further comprising at least three PS bonds connecting the nucleotides in the hairpin 2 region.

[77] The sgRNA according to any one of the above [1] to

[76] , further comprising at least three PS bonds connecting the nucleotides in the upper stem region.

[78] The sgRNA according to any one of the above [1] to

[77] , which forms a ribonucleoprotein complex with Cas9 of S. Pyogenes.

[79] A guide RNA comprising a 2'-O-Me modification at each nucleotide in the upper stem region.

[80] A guide RNA comprising a 2'-O-Me modification in 1, 2, 3, or 4, 5, 6, 7, 8, 9, 10, or 11 of the following nucleotides: US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12.

[81] A guide RNA comprising a 2'-O-Me modification in 50% or more of the nucleotides in the hairpin 1 region and the hairpin 2 region.

[82] A guide RNA comprising a 2'-O-Me modification at each nucleotide in the upper stem region and a 2'-O-Me modification in 50% or more of the nucleotides in the hairpin 1 region and the hairpin 2 region.

[83] A guide RNA comprising or consisting of a 2'-O-Me modification at each nucleotide starting from H2-1 or H2-2 to the last nucleotide at the 3' terminus.

[84] A guide RNA comprising or consisting of a 2'-O-Me modification in LS1 and / or LS6 and no modification in LS2-LS5.

[85] A guide RNA comprising 2'-O-Me modified nucleotides in two or more of LS8, LS9, LS10, LS11, and / or LS12.

[86] The guide RNA according to

[85] above, wherein at least LS8 and LS10 are modified with 2'-O-Me.

[87] The guide RNA according to any one of

[79] -

[86] above, further comprising a 2'-O-Me modification in 50% or more of the nucleotides in the nexus region.

[88] The guide RNA according to

[87] above, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotides in the nexus region are modified with 2'-O-Me.

[89] A guide RNA comprising a 2'-O-Me modification in a maximum of 50% of the nucleotides in the nexus region.

[90] A guide RNA comprising a 2'-O-Me modification in four or more nucleotides in the nexus region.

[91] The guide RNA according to

[90] above, wherein at least one, two, three, four, or five of nucleotides N2 to N6 in the nexus region are modified with 2'-O-Me.

[92] A guide RNA comprising 2'-O-Me modified nucleotides in 10 or more nucleotides in the nexus region.

[93] The guide RNA according to

[92] above, wherein at least N2 to N6 are modified with 2'-O-Me.

[94] A guide RNA comprising 2'-O-Me modification in 50% or more of the nucleotides in the bulge region.

[95] The guide RNA according to

[94] above, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotides in the bulge region are modified with 2'-O-Me.

[96] The guide RNA according to

[94] above, wherein B2, B3, and / or B4 are modified with 2'-O-Me.

[97] A guide RNA comprising 2'-O-Me modified nucleotides in three or more nucleotides in the bulge region.

[98] The guide RNA according to

[97] above, wherein B2, B3, and / or B4 are modified with 2'-O-Me.

[99] A guide RNA comprising the nucleotide of SEQ ID NO: 357 having the modification pattern of the guide RNA according to any one of [1] to

[98] above.

[0100] A guide RNA comprising the nucleotide of SEQ ID NO: 356 having the modification pattern of the guide RNA according to any one of [1] to

[99] above.

[0101] The guide RNA according to any one of

[79] to

[0100] above, further comprising 2'-O-Me modification in the first three, four, or five nucleotides at the 5' end of the 5' terminal region.

[0102] The guide RNA according to any one of

[79] to

[0100] above, further comprising 2'-O-Me modification in the last three, last four, or last five nucleotides at the 3' end of the 3' terminal region.

[0103] The guide RNA according to

[0102] above, wherein the last nucleotide at the 3' end of the 3' terminal region is not modified. The guide RNA according to

[0102] above, wherein only the second last and the third last nucleotides at the 3'-end of the 3'-terminal region are modified. The guide RNA according to

[0102] above, wherein only the second last, the third last, and the fourth last nucleotides in the 3'-terminal region are modified. The guide RNA according to any one of

[79] to

[0100] above, further comprising a PS bond between the first 3, 4, or 5 nucleotides at the 5'-end of the 5'-terminal. The guide RNA according to

[0106] above, wherein the first 4 nucleotides at the 5'-end of the 5'-terminal are linked by a PS bond. The guide RNA according to any one of

[79] to

[0100] above, further comprising a PS bond between the last 3, 4, or 5 nucleotides at the 3'-end of the 3'-terminal. The guide RNA according to

[0108] above, wherein the last 4 nucleotides at the 3'-end of the 3'-terminal are linked by a PS bond. The guide RNA according to any one of

[79] to

[0100] above, wherein the first 3 or 4 nucleotides at the 5'-end of the 5'-terminal and the last 3 or 4 nucleotides at the 3'-end of the 3'-terminal include 2'-O-Me modification. The guide RNA according to any one of

[79] to

[0100] above, wherein the first 3 or 4 nucleotides at the 5'-end of the 5'-terminal are linked by a PS bond, and the last 3 or 4 nucleotides at the 3'-end of the 3'-terminal are linked by a PS bond. The guide RNA according to any one of

[79] to

[0100] above, wherein the first 3 or 4 nucleotides at the 5'-end of the 5'-terminal include 2'-O-Me modification and are linked by a PS bond, and the last 3 or 4 nucleotides at the 3'-end of the 3'-terminal include 2'-O-Me modification and are linked by a PS bond. The guide RNA according to any one of

[79] to

[0100] above, wherein at least two of nucleotides LS8, LS9, LS10, LS11, and LS12 contain a 2'-O-Me modification. The guide RNA according to any one of

[79] to

[0100] above, wherein at least LS8 and LS10 are modified with 2'-O-Me. The guide RNA according to any one of

[79] to

[0100] above, wherein at least 50% of the nucleotides in the nexus region are modified with 2'-O-Me. The guide RNA according to

[0115] above, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotides in the nexus region are modified with 2'-O-Me. The guide RNA according to

[0115] above, wherein at least N2 to N6 are modified with 2'-O-Me. The guide RNA according to any one of

[79] to

[0100] above, wherein at least 10 of the nucleotides in the nexus region are modified with 2'-O-Me. The guide RNA according to

[0115] above, wherein at least N2 to N6 are modified with 2'-O-Me. The guide RNA according to any one of

[79] to

[0100] above, wherein at least 50% of the nucleotides in the bulge region are modified with 2'-O-Me. The guide RNA according to

[0120] above, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotides in the bulge region are modified with 2'-O-Me. The guide RNA according to

[0121] above, wherein B2, B3, and / or B4 are modified with 2'-O-Me. The guide RNA according to any one of

[79] to

[0100] above, wherein at least 3 of the nucleotides in the bulge region are modified with 2'-O-Me. The guide RNA according to

[0123] above, wherein B2, B3, and / or B4 are modified with 2'-O-Me. The guide RNA according to any one of

[79] to

[0124] above, which is an sgRNA. The guide RNA according to any one of

[79] to

[0125] above, which is a dgRNA. The guide RNA according to

[81] or

[82] above, 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. The guide RNA according to any one of

[79] to

[0127] above, which forms a ribonucleoprotein complex with Cas9 of S. Pyogenes. A single guide RNA (sgRNA), a. the first 3 nucleotides at the 5'-end of the 5'-terminus, b. each nucleotide in the upper stem region, c. each nucleotide in the hairpin 1 region, d. the nucleotides between hairpin 1 and hairpin 2, e. each nucleotide in the hairpin 2 region, f. the last 4 nucleotides at the 3'-end of the 3'-terminus, which contains or consists of 2'-O-Me modified nucleotides. The sgRNA according to

[0129] above, further comprising a PS bond connecting the first 4 nucleotides at the 5'-end of the 5'-terminus and a PS bond connecting the last 4 nucleotides at the 3'-end of the 3'-terminus. The sgRNA having a modification pattern shown in SEQ ID NO: 350, 351, 352, or 353 depicted in Table 4. A single guide RNA (sgRNA), a. the first 3, 4, 5, or 7 nucleotides at the 5'-end of the 5'-terminus, b. each nucleotide in the upper stem region, c. each nucleotide in the hairpin 1 region, d. the nucleotides between hairpin 1 and hairpin 2, e. each nucleotide in the hairpin 2 region, f. the last 4 nucleotides at the 3'-end of the 3'-terminus, which contains or consists of 2'-O-Me modified nucleotides. A single guide RNA (sgRNA), a. the first 3, 4, 5, or 7 nucleotides at the 5'-end of the 5'-terminus, b. each of nucleotides US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12, 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, d. nucleotides between hairpin 1 and hairpin 2, 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, f. the last 4 nucleotides at the 3' end of the 3' terminus, wherein the sgRNA comprises or consists of 2'-O-Me modified nucleotides.

[0134] The sgRNA according to any one of

[0129] to

[0133] above, further comprising a PS bond connecting the first 4 nucleotides at the 5' end of the 5' terminus and a PS bond connecting the last 4 nucleotides at the 3' end of the 3' terminus.

[0135] An sgRNA having a modification pattern of SEQ ID NO: 350, 351, 352, or 353 as shown in Table 4.

[0136] The sgRNA according to any one of

[0129] to

[0135] above, which forms a ribonucleoprotein complex with Cas9 of S. Pyogenes.

[0137] A crispr RNA (crRNA), comprising the following regions, namely, a. the first 5 nucleotides at the 5' end of the 5' terminus, b. the lower stem region, c. the bulge region, d. the upper stem region; and e. the last 5 nucleotides at the 3' end of the crRNA, wherein the crRNA comprises one or more modifications in one or more of these regions.

[0138] The crRNA according to

[0136] above, further comprising a 5' end modification, wherein the 5' end modification comprises one or more phosphorothioate linkages within the first 7 nucleotides at the 5' end of the 5' terminus.

[0139] The crRNA according to

[0136] above, further comprising a 5' end modification, wherein the 5' end modification comprises at least two phosphorothioate linkages within the first 7 nucleotides at the 5' end of the 5' terminus.

[0140] The crRNA according to any one of

[0137] to

[0139] above, wherein at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0141] The crRNA according to any one of

[0137] to

[0140] above, wherein at least one modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[0142] The crRNA according to any one of

[0137] to

[0141] above, wherein at least one modification comprises a phosphorothioate (PS) bond between nucleotides.

[0143] The crRNA according to any one of

[0137] to

[0142] above, wherein the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus are modified.

[0144] The crRNA according to any one of

[0137] to

[0143] above, wherein the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus are linked by a phosphorothioate (PS) bond.

[0145] The crRNA according to

[0143] above, wherein the modification comprises 2'-O-Me.

[0146] The crRNA according to

[0143] above, wherein the modification comprises 2'-F.

[0147] The crRNA according to any one of

[0137] to

[0146] above, wherein the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus comprise a 2'-O-Me modification.

[0148] The crRNA according to any one of

[0137] to

[0146] above, wherein the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus comprise a 2'-F modification.

[0149] The crRNA according to any one of

[0137] to

[0148] above, wherein LS1 and LS6 are modified with 2'-O-Me. The crRNA according to any one of

[0137] to

[0149] above, wherein each nucleotide in the upper stem region is modified with 2'-O-Me.

[0151] A crispr RNA (crRNA), a. LS1 and LS6 in the lower stem region, and b. each nucleotide in the upper stem region, the crRNA containing 2'-O-Me modified nucleotides.

[0152] The crRNA according to

[0151] above, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' end of the 3' terminus.

[0153] The crRNA according to

[0151] or

[0152] above, further comprising 2'-O-Me or 2'-F modified nucleotides in the first 3 nucleotides at the 5' end of the 5' terminus and 2'-O-Me or 2'-F modified nucleotides in the last 3 nucleotides at the 3' end of the 3' terminus.

[0154] The crRNA according to any one of

[0137] to

[0153] above, wherein LS1, LS2, and LS6 are modified with 2'-F.

[0155] The crRNA according to any one of

[0137] to

[0154] above, wherein each nucleotide in the bulge region is modified with 2'-F.

[0156] A crispr RNA (crRNA), a. LS1, LS2, and LS6 in the lower stem region, and b. each nucleotide in the bulge region, the crRNA containing 2'-F modified nucleotides.

[0157] The crRNA according to any one of

[0137] to

[0156] above, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' end of the 3' terminus. The crRNA according to any one of

[0137] to

[0157] above, further comprising a 2'-O-Me or 2'-F modified nucleotide in the first 3 nucleotides at the 5'-end of the 5'-terminus, and further comprising a 2'-O-Me or 2'-F modified nucleotide in the last 3 nucleotides at the 3'-end of the 3'-terminus. A crRNA comprising any one of SEQ ID NOs: 1 to 187 including the modifications in Table 4. A crRNA comprising any one of SEQ ID NOs: 19 to 31, 53 to 73, 104 to 130, and 161 to 187 including the modifications in Table 4. 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, and wherein the modification at each nucleotide of the crRNA corresponding to the nucleotide of the reference sequence identifier in Table 4 is the same as or equivalent to the modification shown in the reference sequence identifier in Table 4. The crRNA according to any one of

[0159] to

[0161] above, further comprising three phosphorothioate (PS) bonds linking the first 4 nucleotides at the 5'-end of the 5'-terminus and three PS bonds linking the last 4 nucleotides at the 3'-end of the 3'-terminus. The crRNA according to any one of

[0137] to

[0162] above, wherein the crRNA combined with trRNA forms a ribonucleoprotein complex with Cas9 of S. Pyogenes. A tracr RNA (trRNA), comprising one or more modifications in one or more of the following regions: a. the first 5 nucleotides at the 5'-end of the 5'-terminus; b. the upper stem region; c. the bulge region; d. the lower stem region; e. the nexus region; f. the hairpin 1 region; g. the hairpin 2 region; h. the last 5 nucleotides at the 3'-end of the 3'-terminus. The trRNA according to

[0164] above, wherein at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0166] The tRNA according to the above

[0164] or

[0165] , wherein at least one modification comprises a 2'-fluoro (2'-F) modified nucleotide.

[0167] The tRNA according to any one of the above

[0164] to

[0166] , wherein at least one modification comprises a phosphorothioate (PS) bond between nucleotides.

[0168] The tRNA according to any one of the above

[0164] to

[0167] , wherein the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus are linked by a phosphorothioate (PS) bond.

[0169] The tRNA according to any one of the above

[0164] to

[0168] , wherein the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus are modified.

[0170] The tRNA according to

[0169] above, wherein the modification comprises 2'-O-Me.

[0171] The tRNA according to

[0169] above, wherein the modification comprises 2'-F.

[0172] The tRNA according to any one of the above

[0164] to

[0171] , wherein the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus comprise a 2'-O-Me modification.

[0173] The tRNA according to any one of the above

[0164] to

[0171] , wherein the first 4 nucleotides at the 5' end of the 5' terminus and the last 4 nucleotides at the 3' end of the 3' terminus are linked by a PS bond, and the first 3 nucleotides at the 5' end of the 5' terminus and the last 3 nucleotides at the 3' end of the 3' terminus comprise a 2'-F modification.

[0174] The tRNA according to any one of the above

[0164] to

[0173] , wherein each nucleotide in the upper stem region is modified with 2'-O-Me.

[0175] The tRNA according to any one of the above

[0164] to

[0174] , wherein B1 and B2 in the bulge region are modified with 2'-O-Me. The trRNA according to any one of

[0164] to

[0175] above, wherein N3, N4, N5, N15, N16, N17, and / or N18 in the nexus region are modified with 2'-O-Me. The trRNA according to any one of

[0164] to

[0176] above, wherein each nucleotide in the hairpin 1 region is modified with 2'-O-Me. The trRNA according to any one of

[0164] to

[0177] above, wherein each nucleotide in the hairpin 2 region is modified with 2'-O-Me.

[0179] A tracr RNA (trRNA), a. each nucleotide in the upper stem, b. B1 and / or B2 in the bulge region, c. N3, N4, N5, N15, N16, N17, and / or N18 in the nexus region, d. each nucleotide in the hairpin 1 region, e. each nucleotide in the hairpin 2 region, and which contains 2'-O-Me modified nucleotides, trRNA.

[0180] The trRNA according to

[0179] above, further comprising three phosphorothioate (PS) bonds connecting the first 4 nucleotides at the 5' end of the 5' terminus and three PS bonds connecting the last 4 nucleotides at the 3' end of the 3' terminus.

[0181] The trRNA according to

[0179] or

[0180] above, further comprising 2'-O-Me or 2'-F modified nucleotides in the first 3 nucleotides at the 5' end of the 5' terminus and further comprising 2'-O-Me or 2'-F modified nucleic acids in the last 3 nucleotides at the 3' end of the 3' terminus.

[0182] The trRNA according to any one of

[0164] to

[0181] above, wherein N15, N16, N17, and N18 are modified with 2'-F.

[0183] The trRNA according to any one of

[0164] to

[0182] above, wherein LS1, LS3, and LS5 are modified with 2'-F and LS2, LS4, and LS6 are modified with 2'-O-Me. The trRNA according to any one of

[0164] to

[0183] above, further comprising three phosphorothioate (PS) bonds that link the first 4 nucleotides at the 5'-end of the 5'-terminus and three PS bonds that link the last 4 nucleotides at the 3'-end of the 3'-terminus. The trRNA according to any one of

[0164] to

[0184] above, further comprising a 2'-O-Me or 2'-F modified nucleotide at the first 3 nucleotides at the 5'-end of the 5'-terminus and a 2'-O-Me or 2'-F modified nucleotide at the last 3 nucleotides at the 3'-end of the 3'-terminus. A trRNA comprising any one of SEQ ID NOs: 188 to 227 including the modifications in Table 4. 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 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 as or equivalent to the modification shown in the reference sequence identifier in Table 4. The trRNA according to any one of

[0186] to

[0187] above, further comprising three phosphorothioate (PS) bonds that link the first 4 nucleotides at the 5'-end of the 5'-terminus and three PS bonds that link the last 4 nucleotides at the 3'-end of the 3'-terminus. The trRNA according to any one of

[0164] to

[0188] above, wherein the trRNA combined with crRNA forms a ribonucleoprotein complex with Cas9 of S. Pyogenes. A dual guide comprising crRNA and trRNA, wherein the crRNA comprises any one of SEQ ID NOs: 1 to 187 depicted in Table 4 and the trRNA comprises a nucleic acid of any one of SEQ ID NOs: 188 to 227 depicted in Table 4. A dual guide comprising the crRNA according to any one of

[0137] to

[0163] above and the trRNA according to any one of

[0164] to

[0189] above. A dual guide comprising the crRNA according to any one of

[0137] to

[0163] above and an unmodified trRNA. A dual guide comprising an unmodified crRNA and the trRNA according to any one of

[0137] to

[0189] above. The dual guide according to any one of

[0190] to

[0193] above, which forms a ribonucleoprotein complex with Cas9 of S. Pyogenes. An LNP composition comprising the sgRNA according to any one of [1] to

[78] and

[0129] to

[0136] above. An LNP composition comprising a gRNA comprising or consisting of the RNA according to any one of [1] to

[0193] above. A composition comprising the sgRNA according to any one of [1] to

[78] and

[0129] to

[0136] above, associated with lipid nanoparticles (LNP). A composition comprising the crRNA according to any one of

[0137] to

[0163] above, associated with lipid nanoparticles (LNP). A composition comprising the trRNA according to any one of

[0164] to

[0189] above, associated with lipid nanoparticles (LNP). A composition comprising the sgRNA according to any one of [1] to

[78] and

[0129] to

[0136] above, the gRNA according to any one of

[79] to

[0128] above, the crRNA according to any one of

[0137] to

[0163] above, the trRNA according to any one of

[0164] to

[0189] above, the dgRNA according to any one of

[0190] to

[0194] above, or the composition according to any one of

[0195] to

[0199] above, and further comprising a nuclease or an mRNA encoding the nuclease. The composition according to

[0200] above, wherein the nuclease is a Cas protein. The composition according to

[0201] above, wherein the Cas protein is Cas9. The composition according to

[0202] above, wherein the Cas9 is Cas9 of S. Pyogenes. The composition according to any one of

[0200] to

[0203] above, wherein the nuclease is a nickase. The composition according to any one of

[0200] to

[0204] above, wherein the nuclease is modified. The composition according to

[0205] above, wherein the modified nuclease contains a nuclear localization signal (NLS). The composition according to any one of

[0200] to

[0206] above, which contains mRNA encoding the nuclease. A pharmaceutical preparation comprising the sgRNA according to any one of [1] to

[78] and

[0129] to

[0136] above, the gRNA according to any one of

[79] to

[0128] above, the crRNA according to any one of

[0137] to

[0163] above, the trRNA according to any one of

[0164] to

[0189] above, the dgRNA according to any one of

[0190] to

[0194] above, or the composition according to any one of

[0195] to

[0207] above, and a pharmaceutically acceptable carrier. A method for modifying a target DNA, comprising: A Cas protein or a nucleic acid encoding a Cas protein, and the following: a. The sgRNA according to any one of [1] to

[78] and

[0129] to

[0136] above; b. The gRNA according to any one of

[79] to

[0128] above; c. The crRNA according to any one of

[0137] to

[0163] above; d. The trRNA according to any one of

[0164] to

[0189] above; e. The dgRNA according to any one of

[0190] to

[0194] above; f. The composition according to any one of

[0195] to

[0207] above; or g. Any one or more of the pharmaceutical preparations according to

[0208] above, and delivering the same to a cell. The method according to

[0209] above, which results in an insertion or deletion in a gene. The method according to

[0209] above, further comprising delivering a template to the cell, and at least a part of the template is incorporated into the target DNA at or near the double-strand break site induced by the Cas protein. The sgRNA according to any one of [1] to

[78] and

[0129] to

[0136] above, for use in the preparation of a medicament for treating a disease or disorder. The crRNA according to any one of the above items

[0737] to

[0163] for use in the preparation of a medicament for treating a disease or disorder. The trRNA according to any one of the above items

[0164] to

[0189] for use in the preparation of a medicament for treating a disease or disorder. The crRNA according to any one of the above items

[0137] to

[0163] , in combination with the trRNA according to any one of the above items

[0164] to

[0189] , for use in the preparation of a medicament for treating a disease or disorder. The pharmaceutical preparation according to the above item

[0208] for use in the preparation of a medicament for treating a disease or disorder.

Claims

1. A single guide RNA (sgRNA), comprising: (1) a 5' end comprising a guide region containing 20 nucleotides, and (2) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAACGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 356), where * indicates that the nucleotide is linked to the next nucleotide by a phosphorothioate (PS) bond, and lowercase "m" indicates that the nucleotide is modified with 2'-O-Me, the sgRNA.

2. The sgRNA according to claim 1, wherein at least the first 3 nucleotides at the 5' end of the 5' end are modified.

3. The first 4 nucleotides at the 5' end of the 5' end are linked by a phosphorothioate (PS) bond, and optionally, the sgRNA further comprises a 2'-O-Me or 2'-F modification at the 5' end. The sgRNA according to claim 1 or 2.

4. The first 4 nucleotides at the 5' end of the 5' end are linked by a PS bond, and the first 3 nucleotides at the 5' end of the 5' end comprise a 2'-O-Me modification. The sgRNA according to any one of claims 1 to 3.

5. The first 4 nucleotides at the 5' end are linked by a PS bond, and the first 3 nucleotides at the 5' end comprise one or more of 2'-O-Me, 2'-F, or 2'-O-moe modifications. The sgRNA according to any one of claims 1 to 3.

6. mU*mU*mA*CAGCCACGU CUACAGCAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUA AGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 242), and * indicates that the nucleotide is linked to the next nucleotide by a PS bond, The lowercase letter "m" indicates that the nucleotide is modified with 2'-O-Me, the sgRNA according to claim 1.

7. mN*mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUA AGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 358), and * indicates that the nucleotide is linked to the next nucleotide by a PS bond, The lowercase letter "m" indicates that the nucleotide is modified with 2'-O-Me, N is any nucleotide, the sgRNA according to claim 1.

8. The sgRNA according to any one of claims 1 to 7, which forms a ribonucleoprotein complex with Cas9 of S. Pyogenes.

9. A composition comprising the sgRNA according to any one of claims 1 to 8 associated with a lipid nanoparticle (LNP).

10. A composition comprising the sgRNA according to any one of claims 1 to 8, or the composition according to claim 9, and further comprising a nuclease or an mRNA encoding the nuclease.

11. The composition according to claim 10, wherein the nuclease is a Cas protein.

12. The composition according to claim 10 or 11, wherein the nuclease is Cas9.

13. The composition according to claim 12, wherein the nuclease is Cas9 of S. Pyogenes.

14. The composition according to any one of claims 10 to 13, wherein the nuclease is a nickase or a modified nuclease.

15. The composition according to any one of claims 10 to 14, wherein the nuclease comprises a nuclear localization signal (NLS).

16. A pharmaceutical preparation comprising the sgRNA according to any one of claims 1 to 8, or the composition according to any one of claims 9 to 15, and a pharmaceutically acceptable carrier.

17. An in vitro method for modifying a target DNA, comprising: delivering to a cell a Cas protein or a nucleic acid encoding a Cas protein, and one or more of the following: a. the sgRNA according to any one of claims 1 to 8; b. the composition according to any one of claims 9 to 15; or c. any one or more of the pharmaceutical preparations according to claim 16.

18. A composition comprising the sgRNA according to any one of claims 1 to 8, the composition according to any one of claims 9 to 15, or the pharmaceutical preparation according to claim 16, for use in the treatment of a disease or disorder.

19. A composition comprising the sgRNA according to any one of claims 1 to 8, the composition according to any one of claims 9 to 15, or the pharmaceutical preparation according to claim 16, for use in modifying a target DNA in a cell.

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