Modified CRISPR-Based Gene Editing Systems and Methods of Use

Abstract

The present disclosure discloses a system comprising one or more modified single guide RNAs (sgRNAs) and a donor DNA, wherein each of the modified sgRNAs comprises one or more internal anchors at least 5 nucleotides away from both the 3' and 5' ends of each of the modified sgRNAs, and the donor DNA comprises one or more binding segments capable of binding to one of the one or more internal anchors. The present disclosure also discloses methods of using the systems described herein.

Description

[Technical field]

[0001] (Statement regarding federally sponsored research) This disclosure was made with government support under Grant No. 2016-02830 awarded by the Swedish Research Council to Zongli Zheng.

[0002] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 318,362, filed March 9, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Gene editing technology is rapidly developing and is a powerful tool to manipulate genetic material in target cells, tissues, or organisms. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-related technology is one of the most promising gene editing tools available. However, the low rate of desired precise homology-directed repair (HDR)-mediated editing, the less satisfactory off-target rate, the high translocation rate and the resulting mutagenesis are the main obstacles in the further development and more widespread use of this technology. Summary of the Invention [Means for solving the problem]

[0004] In one embodiment, to address the demand for more effective and specific gene editing results, a system for modifying a target sequence is provided, comprising a modified single guide RNA (sgRNA) and a donor DNA, wherein the modified sgRNA comprises a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA), wherein the modified sgRNA comprises one or more internal anchors at least 5 nucleotides away from both the 3' and 5' ends of the modified sgRNA, and wherein the donor DNA comprises a first portion and a second portion, wherein the first portion comprises one or more binding segments capable of binding to one of the one or more internal anchors via a non-covalent bond, and wherein the second portion comprises a sequence of interest (SOI).

[0005] In some embodiments, the non-covalent bond is a Watson-Crick type interaction.

[0006] In some embodiments, the modified sgRNA comprises a nexus, a first hairpin, and a single-stranded region between the tracrRNA and the crRNA. In some embodiments, the modified sgRNA further comprises a bulge region. In some embodiments, the modified sgRNA further comprises a second hairpin.

[0007] In some embodiments, one of the one or more internal anchors is located in a single-stranded region of the modified sgRNA. In some specific embodiments, one of the one or more internal anchors is located in a single-stranded region between the tracrRNA and the crRNA. In another specific embodiment, one of the one or more internal anchors is located in a single-stranded region in the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in a single-stranded region between the nexus and the first hairpin. In another specific embodiment, the modified sgRNA further comprises a second hairpin, and the single-stranded region is present in the second hairpin.

[0008] In some embodiments, each of the one or more internal anchors or each of the one or more linking segments is between 3 nucleotides and 1000 nucleotides in length. In other embodiments, each of the one or more internal anchors or each of the one or more linking segments is between 3 nucleotides and 20 nucleotides in length. In yet other embodiments, each of the one or more internal anchors or each of the one or more linking segments is about 5 nucleotides in length.

[0009] In some embodiments, the one or more internal anchors each comprise a sequence set forth in SEQ ID NOs: 1-472 of Table 1. In another embodiment, the one or more internal anchors each comprise a sequence set forth in SEQ ID NOs: 473-3056 of Table 2. In another embodiment, the one or more linking segments each comprise a sequence set forth in SEQ ID NOs: 3057-3528 of Table 3. In another embodiment, the one or more linking segments each comprise a sequence set forth in SEQ ID NOs: 3529-6112 of Table 4.

[0010] In some embodiments, one or more binding segments are linked by a linker. In some specific embodiments, the linker is about 1-30 nucleotides in length. In another specific embodiment, the linker is about 10-25 nucleotides in length. In another embodiment, the linker is a polydeoxyadenosine sequence.

[0011] In some embodiments, the SOI comprises a target sequence having one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions, or any combination thereof. In some embodiments, the one or more nucleotide insertions comprise between 1 and 100 nucleotides. In some embodiments, the one or more nucleotide insertions comprise between 101 and 1,000 nucleotides. In some embodiments, the one or more nucleotide insertions comprise between 1,001 and 10,000 nucleotides. In some embodiments, the one or more nucleotide insertions comprise between 1,001 and 10,000 nucleotides. In some embodiments, the one or more nucleotide insertions comprise between 10,001 and 100,000 nucleotides. In another embodiment, the one or more nucleotide insertions comprise between 2 and 10 random nucleotides. In another embodiment, the one or more nucleotide deletions comprise between 1 and 50 nucleotides.

[0012] In some embodiments, the second portion of the donor DNA further comprises an upstream and / or downstream homology arm. In certain embodiments, the upstream homology arm is 5-1000 nucleotides in length. In certain embodiments, the downstream homology arm is about 10-1000 nucleotides in length. In other embodiments, the upstream homology arm is 100-1,000 nucleotides in length. In other embodiments, the downstream homology arm is about 41-1,000 nucleotides in length.

[0013] In some embodiments, the first portion of the donor DNA is located 5' to the second portion of the donor DNA. In other embodiments, the first portion of the donor DNA is located 3' to the second portion of the donor DNA.

[0014] In some embodiments, the donor DNA is single-stranded. In other embodiments, the first portion of the donor DNA is single-stranded and the second portion of the donor DNA is fully or partially double-stranded.

[0015] In some embodiments, the donor DNA is closed at the 3' and / or 5' ends.

[0016] In some embodiments, the system further comprises a CRISPR nuclease. In certain embodiments, the CRISPR nuclease is a DNA nuclease. In certain embodiments, the DNA nuclease is Cas9, Cas12, Cas14 or CasΦ.

[0017] In another embodiment, a system is provided for cleaving a first locus on a first chromosome and a second locus on a second chromosome, comprising a donor DNA and two modified single guide RNAs (sgRNAs), each of said modified sgRNAs comprising a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA), each of said modified sgRNAs comprising one or more internal anchors at least 5 nucleotides away from both a 3' end and a 5' end of each of said modified sgRNAs, said donor DNA comprising a first portion and a second portion, said first portion comprising one or more binding segments capable of binding to one of said one or more internal anchors via a non-covalent bond, said second portion comprising a sequence of interest (SOI), and said donor DNA comprising an upstream homology arm and / or a downstream homology arm.

[0018] In some embodiments, the first chromosome and the second chromosome are the same. In some specific embodiments, the first locus is located 5' of the second locus. In another embodiment, the first chromosome and the second chromosome are different. In some embodiments, the two modified sgRNAs target different loci on the same gene. In some embodiments, the two modified sgRNAs target different loci on the same strand. In some embodiments, the two modified sgRNAs target different loci on different strands. In some embodiments, the system includes a second donor DNA. In some embodiments, each of the donor DNA and the second donor DNA includes an upstream homology arm and / or a downstream homology arm. In some embodiments, the homology arm(s) of the donor DNA and the homology arm(s) of the second donor DNA are complementary to sequences on the same strand. In some embodiments, the homology arm(s) of the donor DNA and the homology arm(s) of the second donor DNA are complementary to sequences on different strands.

[0019] In some embodiments, the first locus and the second locus are separated by at least 50, 100, 1,000, 10,000, or 100,000 nucleotides.

[0020] In some embodiments, the upstream homology arm is adjacent to the 5' end of the first locus. In other embodiments, the downstream homology arm is adjacent to the 3' end of the second locus.

[0021] In some embodiments, the non-covalent bond is a Watson-Crick type interaction.

[0022] In some embodiments, the modified sgRNA comprises a nexus, a first hairpin, and a single-stranded region between the tracrRNA and the crRNA. In some embodiments, the modified sgRNA further comprises a bulge region. In some embodiments, the modified sgRNA further comprises a second hairpin.

[0023] In some embodiments, one of the one or more internal anchors is located in a single-stranded region of the modified sgRNA. In some specific embodiments, one of the one or more internal anchors is located in a single-stranded region between the tracrRNA and the crRNA. In another specific embodiment, one of the one or more internal anchors is located in a single-stranded region in the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in a single-stranded region between the nexus and the first hairpin. In another embodiment, the modified sgRNA further comprises a second hairpin, and the single-stranded region is present in the second hairpin.

[0024] In some embodiments, each of the one or more internal anchors or each of the one or more linking segments is between 3 nucleotides and 100 nucleotides in length. In other embodiments, each of the one or more internal anchors or each of the one or more linking segments is between 3 and 20 nucleotides in length. In yet other embodiments, each of the one or more internal anchors or each of the one or more linking segments is about 5 nucleotides in length.

[0025] In some embodiments, the one or more internal anchors each comprise a sequence set forth in SEQ ID NOs: 1-472 of Table 1. In another embodiment, the one or more internal anchors each comprise a sequence set forth in SEQ ID NOs: 473-3056 of Table 2. In another embodiment, the one or more linking segments each comprise a sequence set forth in SEQ ID NOs: 3057-3528 of Table 3. In another embodiment, the one or more linking segments each comprise a sequence set forth in SEQ ID NOs: 3529-6112 of Table 4.

[0026] In some embodiments, one or more binding segments are linked by a linker. In some specific embodiments, the linker is about 1-30 nucleotides in length. In another specific embodiment, the linker is about 10-25 nucleotides in length. In another embodiment, the linker is a polydeoxyadenosine sequence.

[0027] In some embodiments, the SOI comprises a region between a first locus and a second locus having one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions, or any combination thereof. In some embodiments, the one or more nucleotide insertions comprise 1-100 nucleotides. In some embodiments, the one or more nucleotide insertions comprise 101-1,000 nucleotides. In some embodiments, the one or more nucleotide insertions comprise 1,001-10,000 nucleotides. In some embodiments, the one or more nucleotide insertions comprise 1,001-10,000 nucleotides. In some embodiments, the one or more nucleotide insertions comprise 10,001-100,000 nucleotides. In some embodiments, the one or more nucleotide deletions comprise 1-100 nucleotides. In some embodiments, the one or more nucleotide deletions comprise 101-1,000 nucleotides. In some embodiments, the one or more nucleotide deletions comprise 1,001-10,000 nucleotides. In some embodiments, the one or more nucleotide deletions comprise between 1,001 and 10,000 nucleotides. In some embodiments, the one or more nucleotide deletions comprise between 10,001 and 100,000 nucleotides.

[0028] In certain embodiments, the upstream homology arm is 5 to 1000 nucleotides in length. In certain embodiments, the downstream homology arm is about 10 to 1000 nucleotides in length. In another embodiment, the upstream homology arm is 100 to 1,000 nucleotides in length. In another embodiment, the downstream homology arm is about 41 to 1,000 nucleotides in length.

[0029] In some embodiments, the first portion of the donor DNA is located 5' to the second portion of the donor DNA. In other embodiments, the first portion of the donor DNA is located 3' to the second portion of the donor DNA.

[0030] In some embodiments, the donor DNA is single-stranded. In other embodiments, the first portion of the donor DNA is single-stranded and the second portion of the donor DNA is fully or partially double-stranded.

[0031] In some embodiments, the donor DNA is closed at the 3' and / or 5' ends.

[0032] In some embodiments, the system further comprises a CRISPR nuclease. In certain embodiments, the CRISPR nuclease is a DNA nuclease. In certain embodiments, the DNA nuclease is Cas9, Cas12, Cas14 or CasΦ.

[0033] In another aspect, a method of modifying a cell is provided, the method comprising delivering a system described herein.

[0034] In some embodiments, the delivery includes (a) incubating a CRISPR nuclease and a modified sgRNA to form a ribonucleoprotein (RNP) complex; (b) applying a donor DNA to the RNP complex; and (c) delivering the RNP complex-donor DNA from (b) to the cell. In some specific embodiments, in step (a), the ratio of CRISPR nuclease to modified sgRNA is about 1:0.5 to about 1:10. In another specific embodiment, in step (a), the ratio of CRISPR nuclease to modified sgRNA is about 1:1 to 1:2.

[0035] In another embodiment, the delivery comprises: (a) providing one or more vectors comprising a nucleotide sequence encoding a CRISPR nuclease and a nucleotide sequence encoding a modified gRNA; (b) delivering the one or more vectors in (a) to the cell; and (c) delivering donor DNA to the cell. In a particular embodiment, step (c) is performed about 6-48 hours after step (b) is completed.

[0036] In some embodiments, delivery is achieved by viral vectors, liposomes, lipid nanoparticles, or electroporation.

[0037] In some embodiments, the cell is an immune cell. In certain embodiments, the immune cell is a T cell, a B cell, a NK cell, or a hematopoietic stem cell.

[0038] In some embodiments, the method is performed ex vivo or in vivo. In some embodiments, the method is performed ex vivo or in vivo.

[0039] In some embodiments, the desired editing rate is at least 10%, at least 50%, at least 100%, or at least 200% higher than a similar system that does not include a donor DNA comprising a first portion that binds to a modified sgRNA and / or does not include a modified sgRNA with one or more internal anchors. In another embodiment, the method has an off-target rate that is at least 10%, at least 50%, or at least 100% lower than a similar system that does not include a donor DNA comprising a first portion that binds to a modified sgRNA and / or does not include a modified sgRNA with one or more internal anchors. In another embodiment, the method has a translocation rate, large insertion rate, or large deletion rate that is at least 10%, at least 50%, or at least 100% lower than a similar system that does not include a donor DNA comprising a first portion that binds to a modified sgRNA and / or does not include a modified sgRNA with one or more internal anchors.

[0040] In another aspect, a method for treating a genetic disease is provided, comprising administering to a subject an effective amount of the system described herein. In some embodiments, the SOI comprises a sequence that reverses or alleviates the genetic disease.

[0041] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description. In the following detailed description, only exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is also capable of other different embodiments, and its several details can be modified in various obvious respects, all without departing from the present disclosure. Thus, the drawings and description should be regarded as illustrative in nature, and not restrictive.

[0042] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the specification is intended to supersede and / or take precedence over any such conflicting material.

[0043] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0044] The novel features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "Figures" or "FIG."). The drawings are as follows: [Brief description of the drawings]

[0045] [Figure 1A]Figures 1A-1C show the negligible editing efficiency of the CRISPR Cas9 system employing an RNA-DNA fusion-oligo design (referred to as the "fusion-oligo design") as the single guide. (Figure 1A) Schematic diagram of the fusion-oligo design. Cas9, colored in yellow, is positioned with the target double-stranded DNA, and both strands have a nicked gap, which represents the normal editing result of Cas9. The RNA-DNA fusion oligo contains a 5' portion RNA spacer sequence complementary to the bottom target DNA strand, a middle region containing a trans-activating RNA (tracrRNA) sequence (the RNA portion is also called single-guide RNA [sgRNA], colored in red), and a 3' portion of donor DNA, colored in blue thick line, which serves as a homology-directed repair (HDR; containing an "AAG" insertion for the desired "CTT" insertion into the target DNA) template. [Figure 1B] Figures 1A-1C show the negligible editing efficiency of the CRISPR Cas9 system employing an RNA-DNA fusion oligo (called "fusion oligo") design as a single guide. (Figure 1B) The efficiency of the desired editing result ("CTT" insertion at the target position) by three experimental groups: the previously developed Prime Editor 2 (PE2; a plasmid purchased from AddGene 132775, Andrew V. Anzalone, Nature, 2019 [PMID: 31634902]) as a comparison group, the fusion RNA-DNA guide design (the "test" group containing donor DNAs with lengths of 14, 17, and 20 nt, respectively), and the no-Cas9 control group employing the fusion RNA-DNA guide design (the NC group containing donor DNAs with lengths of 14, 17, 20, and 23 nt, respectively). [Figure 1C] Figures 1A-1C show the negligible editing efficiency of the CRISPR Cas9 system employing an RNA-DNA fusion-oligo design (referred to as "fusion oligo design") as a single guide. (Figure 1C) Shows the efficiency of all edits, including desired ("CTT" insertion at the target position) and undesired (other insertions and deletions) edits, from three experimental groups similar to those in (Figure 1B).

[0046] [Figure 2A] Figures 2A-2C show the gene editing efficiency of the CRISPR Cas9 system, which has Watson-Crick base pairing between the RNA tail located at the end of the sgRNA and the DNA tail located at one end of the donor DNA template (referred to as the "Terminal-Anchor design"). (Figure 2A) Schematic of the Terminal-Anchor design. Cas9, colored in yellow, is positioned with the target double-stranded DNA, and both strands have a nicked gap, which represents the successful editing result of Cas9. The sgRNA, colored in red, contains an extended 3' portion complementary to the donor DNA and a 5' portion of the donor DNA. [Figure 2B] Figures 2A-2C show the gene editing efficiency of the CRISPR Cas9 system with Watson-Crick base pairing between the RNA tail located at the end of the sgRNA and the DNA tail located at one end of the donor DNA template (referred to as "Terminal-Anchor design"). (Figure 2B) Efficiency of the desired editing outcome ("CTT" insertion at the target position) by different experimental groups: control without Cas9 and sgRNA with Terminal-Anchor design (first four samples in the "Negative" group have donor DNA tails with lengths of 3, 7, 10 and 13 bases, respectively); without Cas9 and without sgRNA (fifth to seventh samples in the "Negative" group have donor DNA tails with lengths of 7, 10 and 13 bases, respectively); test groups employing Terminal-Anchor design and with Watson-Crick base pairing with lengths of 0, 3, 5, 7, 10 and 13 bases). [Figure 2C]Figures 2A-2C show the gene editing efficiency of the CRISPR-Cas9 system, which has Watson-Crick base pairing between the RNA tail at the end of the sgRNA and the DNA tail at one end of the donor DNA template (referred to as the "Terminal-Anchor design"). (Figure 2C) The efficiency of all edits, including desired ("CTT" insertion at the target position) and undesired (other insertions and deletions), is shown for the same experimental group as in (Figure 2B).

[0047] [Figure 3A] Figures 3A-3E show various designs of donor DNA and their relative positions to the target and guide DNA. (Figure 3A) Schematic of a CRISPR gene editing system with Watson-Crick base pairing between an RNA sequence located internally to the guide RNA and a DNA portion that is part of the donor DNA template (referred to as an "internal anchor design"). Cas9, colored in yellow, is positioned with the target double-stranded DNA, and both strands have a nicked gap that represents a successful editing result of Cas9. Single guide RNA (sgRNA), colored in red, contains an internal 3' portion that is complementary to a portion of the DNA sequence in the donor DNA. [Figure 3B] Figures 3A-3E show various designs of donor DNA and their relative positions to the target DNA and guide DNA. (Figure 3B) An internal anchor design with a partial double-stranded DNA donor and a single-stranded portion complementary to the interior of the sgRNA. An exemplary donor sequence employing such a design is shown at the bottom of Figure 3B. [Figure 3C] Figures 3A-3E show various designs of donor DNA and their relative positions to the target DNA and guide DNA. (Figure 3C) An internal anchor design with a partially double-stranded DNA donor that is closed and a single-stranded portion that is complementary to the interior of the sgRNA. An exemplary donor sequence employing such a design is shown at the bottom of Figure 3C. [Figure 3D]Figures 3A-3E show various designs of donor DNA and their relative positions to the target DNA and guide DNA. (Figure 3D) An internal anchor design with a partially closed single-stranded DNA donor and a single-stranded portion complementary to the interior of the sgRNA. An exemplary donor sequence employing such a design is shown at the bottom of Figure 3D. [Figure 3E] Figures 3A-3E show various designs of donor DNA and their relative positions to the target DNA and guide DNA. (Figure 3E) An internal anchor design with a hairpin(s) single-stranded DNA donor and a single-stranded portion complementary to the interior of the sgRNA. An exemplary donor sequence employing such a design is shown at the bottom of Figure 3E.

[0048] [Figure 4] Figure 1 shows the effect of different positions of the fragment of donor DNA complementary to the guide RNA (hereafter referred to as "tail") of the internal anchor CR1SPR system on the efficiency of the desired gene editing (in this non-limiting example, "CTT" insertion at the target locus) in HEK293T cells, as measured by next generation sequencing. The CRISPR editing system consists of a guide RNA that is partially complementary to the donor DNA. The complementary sequence within the RNA guide is located inside the guide (internal anchor design). The different designs of the tail of the donor DNA are as follows: "0" indicates that a tailless donor was used, "R" indicates that the tail is located at the 3' end of the donor DNA, "RL" indicates that one DNA tail is located at the 3' end of the donor DNA and the second tail is located at the 5' end of the donor DNA, "L" indicates that one DNA tail is located at the 5' end of the donor DNA, and "L10aL" indicates that the above "L" design is followed by 10 deoxyadenosines and a second "L". The final sample used guide and "L10aL" donor DNA but did not add cas9 as a negative control.

[0049] [Diagram 5]Figure 2 shows the effect of donor tail sequence (matching or not matching the internal anchor sequence of the guide RNA) in the internal anchor CRISPR system with left tail (5' end) on the efficiency of the desired gene editing in HEK293T cells, as measured by next generation sequencing. For the guide, "WT" indicates wild-type guide RNA with no internal anchor (IA) inserted. "LA" indicates guide RNA with an IA inserted, where the tail within the donor DNA binds to the guide RNA. Different designs of the tail of the donor DNA (e.g., "L" and "L10aL") are labeled similarly to Figure 4.

[0050] [Figure 6] Figure 2 shows the effect of Cas9 to guide ratio (1:0.6–1:10) and tail design (labeled "L" and "L10aL" as in Figure 6) on the efficiency of desired ("CTT" insertion at the target locus) and undesired (other insertions / deletions) gene editing using the internal anchor CRISPR system in HEK293T cells, as measured by next generation sequencing.

[0051] [Figure 7] Figure 1 shows the effect of the length of the tail-to-tail inner sequence of donor DNA (0, 5, 10, 15, 20, 25, and 30 deoxyadenosines) on the efficiency of desired ("CTT" insertion at the target locus) and undesired (other insertions / deletions) gene editing using the internal anchor CRISPR system in HEK293T cells, as measured by next-generation sequencing.

[0052] [Figure 8]We show the effect of the length of both the distal (D) and proximal (P) homology arms relative to the "protospacer adjacent motif" (PAM) position on the efficiency of desired ("CTT" insertion at the target locus) and undesired (other insertions / deletions) gene editing using the internal anchor CRISPR system in HEK293T cells, as measured by next-generation sequencing.

[0053] [Figure 9A] Figures 9A-9E show the probability of off-target effects and translocations when utilizing several exemplary configurations employing the "internal anchor design." (Figure 9A) Genome-wide target profiling when targeting HEK3 sites using the internal anchor CRISPR system with different guides (WT and IA), donor DNA tails (0 and L10aL), homology arms (D20P16 and D20P36), and SpCas9 (WT). [Figure 9B] Figures 9A-9E show the probability of off-target effects and translocations when utilizing several exemplary configurations employing the "internal anchor design." (Figure 9B) Genome-wide target profiling when targeting HEK3 sites using the internal anchor CRISPR system with different guides (WT and IA), donor DNA tails (0 and L10aL), homology arms (D20P16 and D20P36), and SpCas9 (HiFi). [Figure 9C] Figures 9A-9E show the probability of off-target effects and translocations when utilizing several exemplary configurations employing the "internal anchor design." (Figure 9C) Genome-wide target profiling when targeting HEK3 sites using the internal anchor CRISPR system with different guides (WT and IA), donor DNA tails (0 and L10aL), homology arms (D20P16 and D20P36), and SpCas9 (eCas9). [Figure 9D]Figures 9A-9E show the probability of off-target effects and translocation when using several exemplary configurations employing the "internal anchor design." (Figure 9D) Number of off-target sites in different experimental condition groups. [Figure 9E] 9A-9E show the probability of off-target effects and translocations when using several exemplary configurations employing the "internal anchor design." (FIG. 9E) Total number of GUIDE-seq reads in experimental conditions.

[0054] [Figure 10A] 10A-10C show designs for deleting approximately 1 kb fragments in HEK3. (Abbreviations: asODN: anchored single-stranded DNA; legRNA: lead-editing guide RNA). [Figure 10B] 10A-10C show designs for deleting approximately 1 kb fragments in HEK3. (Abbreviations: asODN: anchored single-stranded DNA; legRNA: lead-editing guide RNA). [Figure 10C] 10A-10C show designs for deleting approximately 1 kb fragments in HEK3. (Abbreviations: asODN: anchored single-stranded DNA; legRNA: lead-editing guide RNA).

[0055] [Figure 11A] Figures 11A-11C show the deletion results of different asODN-legRNA designs. Figure 11A: PCR amplification products of different designs. The expected size of the deletion product is 288 bp (arrow), and the size of the WT product is 175 bp (arrow). [Figure 11B] Figures 11A-11C show the deletion results with different asODN-legRNA designs. Figure 11B: Band intensity ratio of 288 bp (deleted) to 175 bp (WT) in design A1. [Figure 11C] Figures 11A-11C show the deletion results with different asODN-legRNA designs. Figure 11C: IGV visualization of deletion product alignment on the HEK3 locus.

[0056] [Figure 12A] Figures 12A-12C show the deletion results of different asODN-legRNA designs. Figure 12A: PCR amplification products of different designs. The expected size of the deletion product is 172 bp, and the size of the WT product is 1236 bp. [Figure 12B] Figures 12A-12C show the deletion results with different asODN-legRNA designs. Figure 12B: Band intensity ratio of deletion to WT in design A1, triplicate. [Figure 12C] Figures 12A-12C show the deletion results with different asODN-legRNA designs. Figure 12C: IGV visualization of deletion product alignment on the HPRT1 locus.

[0057] [Figure 13A] 13A-13C show the insertion of a 1734 bp fragment at the GAPDH site. [Figure 13B] 13A-13C show the insertion of a 1734 bp fragment at the GAPDH site. [Figure 13C] 13A-13C show the insertion of a 1734 bp fragment at the GAPDH site. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] This disclosure is based, in part, on the surprising discovery that when designing a donor DNA to be bound to a sgRNA, the location of the effective binding is not random. As shown in the figures and in the examples below, the binding between the donor DNA and the sgRNA is located at least 5 nucleotides away from both the 3' and 5' ends of the modified sgRNA.

[0059] Provided herein is the composition of the system described herein.Disclosed herein are various embodiments of non-covalent linkage, position of internal anchor on modified sgRNA, length and sequence of internal anchor / donor DNA binding segment of modified sgRNA, linker between one or more binding segments on donor DNA, SOI, other features of donor DNA, and Cas protein.Furthermore, provided herein is the method of using the system described herein.

[0060] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It is to be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0061] As used herein, the term "CRISPR / Cas" may refer to a ribonucleoprotein complex, such as a two-component ribonucleoprotein complex that includes a single guide RNA (sgRNA) and a CRISPR-associated (Cas) endonuclease. In some cases, CRISPR / Cas includes more than one component. The term "CRISPR" may refer to clustered regularly interspaced short palindromic repeats and associated systems. CRISPR can be used as an adaptive defense system that allows bacteria and archaea to detect and silence foreign nucleic acids (e.g., those from viruses or plasmids). CRISPR can be adapted for use in a variety of cell types and allows polynucleotide editing in a sequence-specific manner. In some cases, one or more elements of a CRISPR system can be derived from a type I, type II, type III, type IV, type V, or type VI CRISPR system. In a CRISPR type II system, a guide RNA can interact with Cas and direct the nuclease activity of the Cas enzyme to a target region. The target region may contain a "protospacer" and a "protospacer adjacent motif" (PAM), and both domains may be used for activity (e.g., cleavage) mediated by the Cas enzyme. The protospacer may be referred to as a target site (or genomic target site). The sgRNA may pair (or hybridize) with the opposite strand of the protospacer (binding site) to guide the Cas enzyme to the target region. The PAM site may refer to a short sequence that is recognized by the Cas enzyme and, in some cases, required for the activity of the Cas enzyme. The sequence and number of nucleotides of the PAM site may vary depending on the type of Cas enzyme.

[0062] As used herein, the term "Cas" may generally refer to a wild-type Cas protein, a fragment thereof, or a mutant or variant thereof. The terms "Cas", "Cas enzyme", "CRISPR enzyme", "CRISPR protein", or "Cas protein" may be used interchangeably throughout this disclosure.

[0063] Cas proteins may include proteins of or derived from CRISPR / Cas type I, II, III or IV systems with RNA-guided polynucleotide binding or nuclease activity. Examples of suitable Cas proteins include CasX, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (also known as Csnl and Csxl2), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (also known as ...X, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, CasX, Cas Cas proteins include CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, homologs thereof, and modified versions thereof. In some cases, the Cas protein may include a protein of or derived from a CRISPR / Cas type V or type VI system, and modified versions thereof. In some cases, the Cas protein may be a catalytically dead or inactive Cas (dCas). In some cases, the Cas protein may have reduced or minimized nuclease activity (i.e., inactivated Cas, or dCas). In some cases, the Cas protein may be functionally linked to one or more additional proteins, such as a nucleic acid polymerase. In one example, the Cas protein may be dCas fused to a reverse transcriptase.

[0064] As used herein, the term "single guide RNA" or "sgRNA" may refer to an RNA molecule (or collectively a group of RNA molecules) that can bind to a Cas protein and serve to target the Cas protein to a specific location within a target polynucleotide (e.g., DNA). A single guide RNA may include a CRISPR RNA (crRNA) segment and a trans-activating crRNA (tracrRNA) segment. As used herein, the term "crRNA" or "crRNA segment" may refer to an RNA molecule or portion thereof that includes a polynucleotide targeting guide sequence, a stem sequence, and optionally a 5'-overhang sequence. The term "tracrRNA" or "tracrRNA segment" may refer to an RNA molecule or portion thereof that includes a protein-binding segment (e.g., a protein-binding segment that can interact with a CRISPR-associated protein, such as Cas9).

[0065] The terms "polynucleotide" or "nucleic acid", as used interchangeably herein, may refer to a polymeric form of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), guide RNA (gRNA), messenger RNA (mRNA), DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. As used herein, the term "oligonucleotide" may refer to a polynucleotide of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA or RNA. The length of a nucleic acid may be expressed with reference to the number of bases in a nucleic acid sequence. For example, a sequence of 100 nucleotides is said to be 100 bases in length. However, for purposes of this disclosure, there may be no upper limit on the length of an oligonucleotide. In some cases, oligonucleotides are known as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" can include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides. Examples of DNA nucleotides include cytosine (C), guanine (G), adenine (A), thymine (T), or modified versions thereof. Examples of RNA nucleotides include C, G, A, uracil (U), or modified versions thereof.

[0066] A "subject" as disclosed herein includes any animal. Thus, in some embodiments, the subject is a mammal, a bird, a reptile, an amphibian, a fish, a plant, a fungus, or a bacterium. Mammalian subjects include, but are not limited to, humans, non-human primates (e.g., gorillas, monkeys, baboons, chimpanzees, etc.), dogs, cats, goats, horses, pigs, cows, sheep, etc., and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, etc.). Avian subjects include, but are not limited to, chickens, ducks, turkeys, geese, quail, pheasants, and birds kept as pets. In some embodiments, suitable subjects include both males and females, and subjects of any age, including embryonic (e.g., in utero or in ovo), infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a human.

[0067] "Treat" or "treatment" can refer to both therapeutic treatment and preventative measures aimed at preventing or delaying (alleviating) the target pathological condition or disorder. Those in need of treatment can include those who already suffer from the disorder, as well as those who are susceptible to the disorder or those who should be prevented from the disorder. For example, a subject can be successfully "treated" for a disease caused by a gain-of-function mutation if, after being administered a therapeutic amount of a composition according to the method of the present disclosure, it shows some degree of alleviation of one or more of the symptoms associated with the particular disease, reduction in morbidity and / or mortality, and one or more observable and / or measurable reduction or absence of improvement in quality of life issues.

[0068] In this specification, certain ranges are indicated by numerical values ​​preceded by the term "about". The term "about" may be used herein to provide literal support for the exact number it precedes, and for numbers near or approximately the number it precedes. When determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context in which it is recited, provides a substantial equivalent to the specifically recited number. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range (to the tenth of the lower limit unless the context clearly dictates otherwise) and any other specified or intervening value within that specified range is included within the scope of the methods and compositions described herein. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are included within the scope of the methods and compositions described herein, subject to any specifically excluded limit in that specified range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions described herein.

[0069] Whenever the terms "at least," "greater than," or "greater than or equal to" precede a first number in a series of two or more numerical values, the terms "at least," "greater than," or "greater than or equal to" apply to each and every numerical value in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0070] Whenever the terms "not more than," "less than," or "less than or equal to" precede a first value in a series of two or more values, the terms "not more than," "less than," or "less than or equal to" apply to each and every value in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0071] As used herein, the term "desired editing efficiency" or "desired editing percentage" refers to the expected editing result with the designed sequence and the designed position based on the SOI design in the donor DNA described herein. In some embodiments, where the SOI comprises a target sequence with one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions, or any combination thereof, the desired editing efficiency or the desired editing percentage is defined as the percentage of the corresponding gene editing product that comprises the target sequence with one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions, or any combination thereof, and if the second part of the donor DNA has any homology arms, is located at the expected locus based on the design of the upstream and / or downstream homology arms.

[0072] The term "upstream homology arm" as used herein refers to a segment of a target sequence or a region between a first locus and a second locus located 5' to the cleavage site. The term "downstream homology arm" as used herein refers to a segment of a target sequence or a region between a first locus and a second locus located 3' to the cleavage site. The term "distal / proximal homology arm" as used herein refers to a segment of a target sequence or a region between a first locus and a second locus located distal or proximal to a protospacer adjacent motif (PAM) site.

[0073] 1. Compositions of the Systems Described Herein In one embodiment, a system for altering a target sequence is provided, comprising a modified single guide RNA (sgRNA) and a donor DNA, wherein the modified sgRNA comprises a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA), wherein the modified sgRNA comprises one or more internal anchors at least 5 nucleotides away from both a 3' end and a 5' end of the modified sgRNA, and wherein the donor DNA comprises a first portion and a second portion, wherein the first portion comprises one or more binding segments capable of binding to one of the one or more internal anchors via a non-covalent bond, and wherein the second portion comprises a sequence of interest (SOI). In another embodiment, a system is provided for cleaving a first locus on a first chromosome and a second locus on a second chromosome, comprising a donor DNA and two modified single guide RNAs (sgRNAs), each of said modified sgRNAs comprising a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA), each of said modified sgRNAs comprising one or more internal anchors at least 5 nucleotides away from both a 3' end and a 5' end of each of said modified sgRNAs, said donor DNA comprising a first portion and a second portion, said first portion comprising one or more binding segments capable of binding to one of said one or more internal anchors via a non-covalent bond, said second portion comprising a sequence of interest (SOI), and said donor DNA comprising an upstream homology arm and / or a downstream homology arm.

[0074] (a) Non-covalent bond In some embodiments, the non-covalent bonds described herein are hydrogen bonds. In certain embodiments, the non-covalent bonds described herein are Watson-Crick type interactions. In another embodiment, the non-covalent bonds described herein are ionic interactions. In another embodiment, the non-covalent bonds described herein are van der Waals interactions. In another embodiment, the non-covalent bonds described herein are hydrophobic bonds.

[0075] (b) Position of the internal anchor on the modified sgRNA In some embodiments, the modified sgRNA comprises a nexus, a first hairpin, and a single-stranded region between the tracrRNA and the crRNA. Thus, in some specific embodiments, one of the one or more internal anchors is located in the single-stranded region between the tracrRNA and the crRNA. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region between the nexus and the first hairpin. In another embodiment, one of the one or more internal anchors is located in the stem portion of the first hairpin, thereby forming an artificial bulge-like structure in the stem portion of the first hairpin. In another specific embodiment, one or more sequences in the tracrRNA are reverse complementary to the corresponding sequences in the crRNA, thereby forming a stem between the tracrRNA and the crRNA. Thus, one of the one or more internal anchors is located in the stem between the tracrRNA and the crRNA, thereby forming an artificial bulge-like structure in the stem.

[0076] In some embodiments, the modified sgRNA comprises a nexus, a first hairpin, a second hairpin, optional one or more hairpins, and a single-stranded region between the tracrRNA and the crRNA. Thus, in some specific embodiments, one of the one or more internal anchors is located in the single-stranded region between the tracrRNA and the crRNA. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the second hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the optional one or more hairpins. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region between the nexus and the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region between the first hairpin and the second hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region between the second and third hairpins, or in the single-stranded region between two adjacent hairpins. In another specific embodiment, one of the one or more internal anchors is located in the stem portion of the first hairpin, thereby forming an artificial bulge-like structure in the stem portion of the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the stem portion of the second hairpin, thereby forming an artificial bulge-like structure in the stem portion of the second hairpin. In another specific embodiment, one of the one or more internal anchors is located in the stem portion of the optional one or more hairpins, thereby forming an artificial bulge-like structure in the stem portion of the optional one or more hairpins. In another specific embodiment, one or more sequences in the tracrRNA are reverse complementary to the corresponding sequences in the crRNA, thus forming a stem between the tracrRNA and the crRNA.This positions one of the one or more internal anchors within the stem between the tracrRNA and the crRNA, thereby forming an artificial bulge-like structure within the stem.

[0077] In some embodiments, the modified sgRNA comprises a nexus, a bulge region, a first hairpin, and a single-stranded region between the tracrRNA and the crRNA. Specifically, one or more sequences in the tracrRNA are reverse-complementary to the corresponding sequences in the crRNA, so that a stem is formed between the tracrRNA and the crRNA. The stem is divided into an upper stem and a lower stem by the bulge region. Thus, in some specific embodiments, one of the one or more internal anchors is located in the single-stranded region between the tracrRNA and the crRNA, and this single-stranded region corresponds to the upper loop of the upper stem. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region between the nexus and the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the stem portion of the first hairpin, thereby forming an artificial bulge-like structure in the stem portion of the first hairpin. In another specific embodiment, one of the one or more internal anchors is located within the upper stem, thereby forming an artificial bulge-like structure in the upper stem. In another specific embodiment, one of the one or more internal anchors is located within the lower stem, thereby forming an artificial bulge-like structure in the lower stem.

[0078] In some embodiments, the modified sgRNA comprises a nexus, a bulge region, a first hairpin, a second hairpin, optional one or more hairpins, and a single-stranded region between the tracrRNA and the crRNA. Specifically, one or more sequences in the tracrRNA are reverse-complementary to the corresponding sequences in the crRNA, so that a stem is formed between the tracrRNA and the crRNA. The stem is divided into an upper stem and a lower stem by the bulge region. Thus, in some specific embodiments, one of the one or more internal anchors is located in the single-stranded region between the tracrRNA and the crRNA, and this single-stranded region corresponds to the upper loop of the upper stem. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the second hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region in the optional one or more hairpins. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region between the nexus and the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the single-stranded region between the first hairpin and the second hairpin, or between any adjacent hairpins. In another specific embodiment, one of the one or more internal anchors is located in the stem portion of the first hairpin, thereby forming an artificial bulge-like structure in the stem portion of the first hairpin. In another specific embodiment, one of the one or more internal anchors is located in the stem portion of the second hairpin, thereby forming an artificial bulge-like structure in the stem portion of the second hairpin. In another specific embodiment, one of the one or more internal anchors is located in the stem portion of the optional one or more hairpins, thereby forming an artificial bulge-like structure in the stem portion of the one or more hairpins.In another specific embodiment, one of the one or more internal anchors is located within the upper stem, thereby forming an artificial bulge-like structure in the upper stem. In another specific embodiment, one of the one or more internal anchors is located within the lower stem, thereby forming an artificial bulge-like structure in the lower stem.

[0079] (c) Length of the internal anchor / donor DNA binding segment of the modified sgRNA In some embodiments, each of the one or more internal anchors is between 3 nucleotides and 100 nucleotides in length. In another embodiment, each of the one or more internal anchors is between 3 nucleotides and 20 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 3 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 4 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 5 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 6 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 7 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 8 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 9 nucleotides in length. In another embodiment, each of the one or more internal anchors is at least 10 nucleotides in length. In another embodiment, each of the one or more internal anchors is 3 nucleotides in length. In another embodiment, each of the one or more internal anchors is 4 nucleotides in length. In another embodiment, each of the one or more internal anchors is 5 nucleotides in length. In another embodiment, each of the one or more internal anchors is 6 nucleotides in length. In another embodiment, each of the one or more internal anchors is 7 nucleotides in length. In another embodiment, each of the one or more internal anchors is 8 nucleotides in length. In another embodiment, each of the one or more internal anchors is 9 nucleotides in length. In another embodiment, each of the one or more internal anchors is 10 nucleotides in length.

[0080] In some embodiments, the binding segment of the donor DNA binds to the internal anchor of the modified sgRNA via Watson-Crick type interactions. Thus, the binding segment of the donor DNA is also approximately the same length as the internal anchor of the modified sgRNA. Thus, in some embodiments, each of the one or more binding segments is 3 nucleotides to 100 nucleotides long. In another embodiment, each of the one or more binding segments is 3 nucleotides to 20 nucleotides long. In another embodiment, each of the one or more binding segments is at least 3 nucleotides long. In another embodiment, each of the one or more binding segments is at least 4 nucleotides long. In another embodiment, each of the one or more binding segments is at least 5 nucleotides long. In another embodiment, each of the one or more binding segments is at least 6 nucleotides long. In another embodiment, each of the one or more binding segments is at least 7 nucleotides long. In another embodiment, each of the one or more binding segments is at least 8 nucleotides long. In another embodiment, each of the one or more binding segments is at least 9 nucleotides long. In another embodiment, each of the one or more binding segments is at least 10 nucleotides long. In another embodiment, each of the one or more binding segments is 3 nucleotides long. In another embodiment, each of the one or more binding segments is 4 nucleotides in length. In another embodiment, each of the one or more binding segments is 5 nucleotides in length. In another embodiment, each of the one or more binding segments is 6 nucleotides in length. In another embodiment, each of the one or more binding segments is 7 nucleotides in length. In another embodiment, each of the one or more binding segments is 8 nucleotides in length. In another embodiment, each of the one or more binding segments is 9 nucleotides in length. In another embodiment, each of the one or more binding segments is 10 nucleotides in length.

[0081] (d) Sequence of the internal anchor / donor DNA binding segment of the modified sgRNA In some embodiments, each of the internal anchors of the modified sgRNA comprises a sequence that is an uncommon motif in the host genome. In certain embodiments, in some embodiments, each of the internal anchors of the modified sgRNA comprises a sequence that is an uncommon motif in the human genome. As a non-limiting example, in some embodiments, each of the internal anchors of the modified sgRNA comprises a sequence in Table 1. In another embodiment, each of the internal anchors of the modified sgRNA comprises a sequence in Table 2. In some embodiments, the binding segment of the donor DNA binds to the internal anchor of the modified sgRNA via Watson-Crick type interactions. Thus, in some embodiments, each of the binding segments of the donor DNA comprises a sequence that is reverse complementary to a sequence in Table 1. In another embodiment, each of the binding segments of the donor DNA comprises a sequence that is reverse complementary to a sequence in Table 2.

[0082] In some embodiments, each of the binding segments of the donor DNA comprises a sequence that is an uncommon motif in the host genome. In certain embodiments, in some embodiments, each of the binding segments of the donor DNA comprises a sequence that is an uncommon motif in the human genome. As a non-limiting example, in some embodiments, each of the binding segments of the donor DNA comprises a sequence in Table 3. In another embodiment, each of the binding segments of the donor DNA comprises a sequence in Table 4. In some embodiments, the binding segments of the donor DNA bind to the internal anchors of the modified sgRNA via Watson-Crick type interactions. Thus, in some embodiments, each of the internal anchors of the modified sgRNA comprises a sequence that is reverse complementary to a sequence in Table 3. In some embodiments, each of the internal anchors of the modified sgRNA comprises a sequence that is reverse complementary to a sequence in Table 4. Table 1. Exemplary sequences of internal anchors of modified sgRNAs (5 nucleotides long) [Table 1-1] [Table 1-2] [Table 1-3] Table 2. Exemplary sequences of internal anchors of modified sgRNAs (6 nucleotides long) [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] Table 3. Exemplary sequences of the binding segment of donor DNA (5 nucleotides long) [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] Table 4. Exemplary sequences of the binding segment of donor DNA (6 nucleotides long) [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13]

[0083] (e) a linker between one or more binding segments on the donor DNA; In some embodiments, a nucleotide sequence (linker) is designed to be present between one or more linking segments on the donor DNA.

[0084] In some embodiments, the linker is about 1-30 nucleotides in length. In another embodiment, the linker is about 10-25 nucleotides in length. In some embodiments, the linker is at least 5 nucleotides in length. In some embodiments, the linker is at least 10 nucleotides in length. In some embodiments, the linker is at least 15 nucleotides in length. In some embodiments, the linker is at least 20 nucleotides in length. In some embodiments, the linker is at least 25 nucleotides in length. In some embodiments, the linker is about 5 nucleotides in length. In some embodiments, the linker is about 10 nucleotides in length. In some embodiments, the linker is about 15 nucleotides in length. In some embodiments, the linker is about 20 nucleotides in length. In some embodiments, the linker is about 25 nucleotides in length. In some embodiments, the linker is about 30 nucleotides in length.

[0085] In some embodiments, the linker is a deoxyadenosine sequence, a deoxyguanosine sequence, a thymidine sequence, a deoxycytidine sequence, or any combination thereof. In some specific embodiments, the linker is a polydeoxyadenosine sequence. In another specific embodiment, the linker is a polythymidine sequence. In another specific embodiment, the linker is a deoxyadenosine and thymidine sequence.

[0086] (f) Sequence of interest (SOI) In some embodiments, the SOI of the donor DNA described herein comprises a target sequence having one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions, or any combination thereof.

[0087] In some specific embodiments, the SOI of the donor DNA described herein comprises a target sequence with a nucleotide insertion of 1-100 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence with a nucleotide insertion of 1-50 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence with a nucleotide insertion of 2-10 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence with a nucleotide insertion of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence with a nucleotide insertion of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence having a nucleotide insertion of at least 50, at least 100, at least 1,000, at least 10,000, or at least 100,000 nucleotides.

[0088] In some specific embodiments, the SOI of the donor DNA described herein comprises a target sequence having a nucleotide deletion of 1-50 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence having a nucleotide deletion of 1-10 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence having a nucleotide deletion of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence having a nucleotide deletion of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides.

[0089] In some specific embodiments, the SOI of the donor DNA described herein comprises a target sequence having nucleotide substitutions of 1-50 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence having nucleotide substitutions of 1-10 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence having nucleotide substitutions of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. In another specific embodiment, the SOI of the donor DNA described herein comprises a target sequence having nucleotide substitutions of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides.

[0090] Additionally, since the SOI of the donor DNA described herein serves as a template for homologous recombination following guide RNA-mediated nuclease cleavage, in some embodiments the second portion of the donor DNA further comprises upstream and / or downstream homology arms.

[0091] In some embodiments, the upstream homology arms described herein are about 10-1,000 nucleotides in length. In another embodiment, the upstream homology arms described herein are about 10-80 nucleotides in length. In some embodiments, the upstream homology arms described herein are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 bases in length. In some specific embodiments, the upstream homology arms described herein are about 20 nucleotides in length. In some specific embodiments, the upstream homology arms described herein are about 25 nucleotides in length. In some specific embodiments, the upstream homology arms described herein are about 30 nucleotides in length. In some specific embodiments, the upstream homology arms described herein are about 35 nucleotides in length.

[0092] In some embodiments, the downstream homology arms described herein are about 10-1,000 nucleotides in length. In another embodiment, the downstream homology arms described herein are about 10-80 nucleotides in length. In some embodiments, the downstream homology arms described herein are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 bases in length. In some specific embodiments, the downstream homology arms described herein are about 20 nucleotides in length. In some specific embodiments, the downstream homology arms described herein are about 25 nucleotides in length. In some specific embodiments, the downstream homology arms described herein are about 30 nucleotides in length. In some specific embodiments, the downstream homology arms described herein are about 35 nucleotides in length.

[0093] In some embodiments, the second portion of the donor DNA further comprises a distal and / or proximal homology arm. In some embodiments, the distal homology arm is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 nucleotides in length. In some embodiments, the proximal homology arm is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 nucleotides in length.

[0094] In some specific embodiments, the SOI of donor DNA described herein is optimized to avoid any PAM sequence of the CRISPR nuclease used in the system.For example, in some embodiments, the SOI of donor DNA described herein is optimized by silent mutation to avoid any PAM sequence of the CRISPR nuclease used in the system, but does not cause any change in the amino acid content in SOI.

[0095] (g) Other characteristics of the donor DNA As shown in Figures 3A-3E, the donor DNA described herein may adopt various designs, as non-limiting examples. In some embodiments, the donor DNA is single-stranded. In another embodiment, the first portion of the donor DNA is single-stranded and the second portion of the donor DNA is fully double-stranded. In another embodiment, the first portion of the donor DNA is single-stranded and the second portion of the donor DNA is partially double-stranded. In some embodiments, the donor DNA is closed at its 3' end. In some embodiments, the donor DNA is closed at its 5' end. In some embodiments, the donor DNA is closed at its 3' and 5' ends. In some particular embodiments, the donor DNA forms a hairpin secondary structure, with one or more binding segments located within the loops of the hairpin. In some particular embodiments, the donor DNA forms a dumbbell-shaped secondary structure, with one or more binding segments located within one loop of the dumbbell. In some particular embodiments, the donor DNA described herein is a circular DNA. In certain embodiments, the donor DNA described herein forms a partial dumbbell-shaped secondary structure, with one or more binding segments located within one loop of the dumbbell.

[0096] Additionally, the first portion of the donor DNA described herein, which includes one or more binding segments, can be designed to be at various locations in the donor DNA, as examined in Figures 4 and 5. In some embodiments, the first portion of the donor DNA is located 5' of the second portion of the donor DNA. In other embodiments, the first portion of the donor DNA is located 3' of the second portion of the donor DNA.

[0097] (h) Cas protein In some embodiments, the systems described herein further comprise a CRISPR nuclease. In some particular embodiments, the CRISPR nuclease is a DNA nuclease.

[0098] In some embodiments, the CRISPR nuclease is a class I CRISPR nuclease. In other embodiments, the CRISPR nuclease is a class II CRISPR nuclease.

[0099] In some embodiments, the CRISPR nuclease is a type I CRISPR nuclease. In some particular embodiments, the CRISPR nuclease is a type IA, type IB, type IC, type ID, type IE, type IF, or type IU CRISPR nuclease. In another embodiment, the CRISPR nuclease is a type II CRISPR nuclease. In some particular embodiments, the CRISPR nuclease is a type II-A, type II-B, or type II-C CRISPR nuclease. In some embodiments, the CRISPR nuclease is a type III CRISPR nuclease. In some particular embodiments, the CRISPR nuclease is a type III-A, type III-B, type III-C, or type III-D CRISPR nuclease. In some embodiments, the CRISPR nuclease is a type IV CRISPR nuclease. In another embodiment, the CRISPR nuclease is a type V CRISPR nuclease. In certain embodiments, the CRISPR nuclease is a type VA, type VB, or type VC CRISPR nuclease.

[0100] In some embodiments, the DNA nuclease described herein is Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas14, CasΦ, Casm, or Cmr. In some specific embodiments, when the DNA nuclease is Cas9, the DNA nuclease is high-fidelity Cas9 or eCas9. In some specific embodiments, when the DNA nuclease is Cas12, the DNA nuclease is Cas12a (Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, or Cas12k.

[0101] In one aspect, a kit is provided that comprises the donor DNA described herein and the modified sgRNA described herein.In some embodiments, the kit comprises the donor DNA described herein, the modified sgRNA described herein, and the CRISPR nuclease described herein.

[0102] In one aspect, a pharmaceutical composition is provided that includes the donor DNA described herein, the modified sgRNA described herein, and a pharma- ceutically acceptable salt or derivative thereof. In some embodiments, the kit includes the donor DNA described herein, the modified sgRNA described herein, and the CRISPR nuclease described herein, and a pharma-ceutically acceptable salt or derivative thereof. 2. Methods of Using the Systems Described Herein

[0103] In one aspect, a method of modifying a cell is provided, the method comprising delivering a system described herein into the cell.

[0104] In some embodiments, the delivery includes (a) incubating a CRISPR nuclease described herein and a modified sgRNA described herein to form a ribonucleoprotein (RNP) complex; (b) applying donor DNA to the RNP complex; and (c) delivering the RNP complex-donor DNA from (b) to the cell. In some specific embodiments, the RNP complex is formed in vitro in step (a). In some embodiments, the CRISPR nuclease described herein is expressed and purified using an associated plasmid. In some embodiments, the modified sgRNA is transcribed in vitro from the corresponding ssDNA. In some non-limiting and exemplary embodiments, in step (a), the ratio of CRISPR nuclease to modified sgRNA is about 1:0.5 to about 1:10. In another non-limiting and exemplary embodiment, in step (a), the ratio of CRISPR nuclease to modified sgRNA is about 1:1 to about 1:1.2. In another non-limiting exemplary embodiment, in step (a), the ratio of CRISPR nuclease to modified sgRNA is about 1:0.6, about 1:1.2, about 1:2, or about 1:5. In some specific embodiments, applying donor DNA to the RNP complex is performed in vitro in step (b). In some specific embodiments, delivering the RNP complex and donor DNA from (b) to a cell is achieved by a viral vector, a liposome, and / or a lipid nanoparticle. In another specific embodiment, delivering the RNP complex and donor DNA from (b) to a cell is achieved by electroporation. In one embodiment, delivering the RNP complex and donor DNA from (b) to a cell is achieved by nucleofection (see, e.g., Distler et al., Exp Dermatol 2005 Apr; 14(4):315-20). In another specific embodiment, delivering the RNP complex and donor DNA from (b) to a cell is achieved by polyethylene glycol (PEG)-mediated transfection.In another specific embodiment, delivering the RNP complex-donor DNA from (b) to the cell is accomplished by a gene gun.

[0105] In some embodiments, the delivery comprises (a) providing one or more vectors comprising a nucleotide sequence encoding a CRISPR nuclease described herein and a nucleotide sequence encoding a modified gRNA described herein; (b) delivering one or more vectors in (a) to the cell; and (c) delivering a donor DNA described herein to the cell. In some preferred embodiments, step (c) is performed about 6 to 48 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 6 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 12 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 18 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 24 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 30 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 36 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 42 hours after step (b) is completed. In another preferred embodiment, step (c) is performed at least 48 hours after step (b) is completed. In some specific embodiments, delivering one or more vectors in (a) to the cell is achieved by a viral vector, a liposome, and / or a lipid nanoparticle. In some specific embodiments, delivering donor DNA to the cell is achieved by a viral vector, a liposome, and / or a lipid nanoparticle.

[0106] In some embodiments, the modified cell is an immune cell. In some specific embodiments, the modified cell is a T cell. In some specific embodiments, the modified cell is a B cell. In some specific embodiments, the modified cell is a NK cell. In some specific embodiments, the modified cell is a hematopoietic stem cell.

[0107] In some embodiments, the methods described herein are performed in vitro. In other embodiments, the methods described herein are performed ex vivo. In other embodiments, the methods described herein are performed in vivo.

[0108] Due to the relatively high cleavage efficiency of the nucleases described herein and the relatively low efficiency of homology-directed repair (HDR), the majority of nuclease-induced double-strand breaks (DSBs) can be repaired by NHEJ (Non-Homologous End Joining). In other words, the resulting cell population can contain some combination of wild-type alleles, NHEJ repair alleles, and / or desired edited alleles based on the design of the SOI of the donor DNA described herein. The method of modifying cells using the system described herein is advantageous in terms of a high rate of desired editing, a low off-target rate, and / or a low translocation rate, a large insertion rate, or a large deletion rate.

[0109] In some embodiments, the rate of desired editing using the methods described herein is at least 10%, at least 50%, at least 100%, or at least 200% higher than a similar system that does not include a donor DNA comprising a first moiety that binds to a modified sgRNA and / or does not include a modified sgRNA. In another embodiment, the methods described herein have an off-target rate that is at least 10%, at least 50%, or at least 100% lower than a similar system that does not include a donor DNA comprising a first moiety that binds to a modified sgRNA and / or does not include a modified sgRNA. In another embodiment, the methods have a translocation rate, large insertion rate, or large deletion rate that is at least 10%, at least 50%, or at least 100% lower than a similar system that does not include a donor DNA comprising a first moiety that binds to a modified sgRNA and / or does not include a modified sgRNA.

[0110] To further increase the rate of HDR, in some embodiments, the method described herein further comprises synchronizing cells in S phase. In another embodiment, the method described herein further comprises synchronizing cells in G2 phase. In another embodiment, the method described herein further comprises inhibiting a gene involved in the NHEJ pathway. In another embodiment, the method described herein further comprises inhibiting a gene involved in the mismatch repair pathway. In another embodiment, the method described herein further comprises fusing ctIP, a protein involved in double-stranded break resection, to CRISPR nuclease. In another embodiment, the method described herein further comprises fusing single-stranded annealing protein to CRISPR nuclease.

[0111] In another aspect, a method for treating a genetic disease is provided, comprising administering to a subject an effective amount of the system described herein. In some embodiments, the SOI comprises a sequence that reverses or alleviates the genetic disease. Working Example

[0112] The following are descriptions of various methods and materials used in the study. These are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors consider to be their disclosure, nor are they intended to represent all of the experiments that have been performed and may be performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, proportions, etc.), but some experimental error and deviation should be taken into account.

[0113] Example 1: The "fusion oligo design" and "terminal anchor design" proved to be dysfunctional "Fusion oligo design" refers to a system in which donor DNA was fused to guide RNA (see Figure 1A). Briefly, guide RNA-donor DNA fusions with various lengths of donor DNA, listed in Table 5 below, were tested. As shown in Figure 1B and Figure 1C, the "fusion oligo design" showed negligible editing efficiency compared to the positive control. Table 5. Guide RNA-donor DNA fusion sequences [Table 5] Table 6 Donor DNA sequence in "Terminal anchor design" [Table 6]

[0114] Separately, "terminal anchor design" was also tested. "Terminal anchor design" refers to a system that includes a guide RNA that is partially complementary to the donor DNA, and the complementary sequence is located at the end of the guide RNA (see Figure 2A). Briefly, donor DNAs with complementary sequences of different lengths from the 3' end of the guide RNA, as listed in Table 6 above, were tested. As shown in Figure 2B and Figure 2C, "fusion oligo design" did not increase the desired editing efficiency.

[0115] Based on the above surprising and unexpected observations, we concluded that the type and position of the linkage between the donor DNA and the guide RNA substantially affect the editing efficiency. Fusion or hybridization of the ends of the donor DNA and the guide RNA did not result in satisfactory editing efficiency.

[0116] Example 2: Various designs for donor DNA and guide RNA for "internal anchor design" "Internal anchor design" refers to a system that includes a guide RNA that is partially complementary to the donor DNA, where the complementary sequence is located internally within the guide RNA (see Figure 3A). A variety of different donor constructs were also designed (see Figures 3B-3E).

[0117] Using the internal anchor CRISPR system, we first evaluated the effect of different positions of the fragment of donor DNA ("tail") complementary to the guide RNA on the efficiency of the desired gene editing ("CTT" insertion at the target locus) in HEK293T cells. The different DNA donor designs included a conventional design where the donor DNA had no tail (denoted by "0" in Figure 4), a design where the DNA sequence complementary to the guide RNA was located at the 3' end of the donor DNA (denoted by "R" in Figure 4), a design where one DNA tail was located at the 3' end of the donor DNA and a second "tail" was located at the 5' end of the donor DNA (denoted by "RL" in Figure 4), and a design where one DNA tail was located at the 5' end of the donor DNA (denoted by "L" in Figure 4), where the "L" design was followed by 10 deoxyadenosines and a second "L" (denoted by "L10aL" in Figure 4). One situation where no Cas9 was added was included as a negative control (last column in Figure 4). The results showed that the "R" and "RL" designs had lower desired editing efficiencies compared to conventional DNA donors, whereas the "L" and "L10aL" designs had statistically significantly higher desired editing efficiencies compared to conventional DNA donors (see Figure 4).

[0118] We next tested the effect of the tail sequence of the donor DNA, matching or mismatching the internal anchor (IA) sequence of the guide RNA, on the editing efficiency. As expected, the negative control samples (no guide RNA, no donor DNA, or no Cas9, shown as "-" in the categories "guide", "donor tail", or "Cas9") showed no observable CTT insertion (see Figure 5). Furthermore, three other situations showed similar CTT insertion efficiencies, around 5%-6%, including (1) a WT guide RNA without an IA sequence and a donor without a tail ("guide:WT" and "donor tail:0"), (2) a donor with a WT guide RNA without an IA sequence and a mock tail ("guide:WT" and "donor tail:nonM"), and (3) a guide RNA with an IA sequence and a mismatched donor tail containing a mock 5-nucleotide sequence that did not match the guide IA ("guide:IA" and "donor tail:nonM"). In contrast, guide RNAs with IA sequences and donor DNAs employing either the "L" or "L10aL" tail designs ("guide:IA" and "donor tail:L", and "guide:IA" and "donor tail:L10aL") both increased the CTT insertion efficiency by approximately two-fold (see Figure 5).

[0119] Example 3: Guide RNA to Cas9 ratio Two tail designs ("L" and "L10aL", labeled as in Example 1) were employed and different ratios of the two (1:0.6 to 1:10) were tested. As shown in Figure 6, the efficiency of desired (in this non-limiting example, "CTT" insertion at the target locus) and undesired (other insertions / deletions) gene editing showed that guide RNA to Cas9 ratios of 1:1.2 and 1:2 had the highest efficiency compared to other ratios. However, a higher number of RNA guides (ratios of 1:5 or 1:10) significantly reduced the desired editing efficiency, while other insertions / deletions, such as those by the non-homologous DNA end joining (NHEJ) pathway, were not affected (see Figure 6). Thus, the relative amounts of Cas9 and guide RNA affected the editing efficiency.

[0120] Example 4: Length of the internal linker between two binding segments on the donor DNA Using the internal anchor CRISPR system in HEK293T cells, we investigated the effect of donor DNA tail-to-tail inner sequence length (0, 5, 10, 15, 20, 25, and 30 deoxyadenosines, Figure 7) on the efficiency of desired ("CTT" insertion at the target locus) and undesired (other insertions / deletions) gene editing. The length of 10 deoxyadenosines ("L10aL") showed the highest desired CTT insertion compared to other lengths of donor DNA. Correspondingly, the L10aL design showed the lowest rate of undesired other insertions / deletions, indicating that undesired editing by-products via the NHEJ pathway were substantially suppressed by the L10aL design.

[0121] Example 5: Length of Homology Arms The effect of DNA donor homology arm length on HDR editing efficiency was studied. Donors with different lengths distal (D) and proximal (P) to the cut position were tested, as shown in Figure 8. Both short (less than 20 bases) and long (more than 36 bases) homology arms showed lower CTT insertion efficiency compared to donors with intermediate lengths (20-36 bases). For donors with D20P30 and D20P36 homology arms, the percentage of CTT insertion reads was higher than the number of unwanted insertion / deletion reads in the same sample.

[0122] Example 6: Off-target rate, translocation rate, large insertion rate, large deletion rate Genome-wide off-target profiling was evaluated when targeting HEK3 sites using internal anchor CRISPR systems with various elements: (1) different guides ("WT" indicates a guide RNA without an internal anchor and "LA" indicates a guide RNA with an internal anchor), (2) donor DNA tails ("0" indicates donor DNA without a tail / binding segment complementary to the guide RNA, and "L10aL" indicates that two DNA tails / binding segments are located at the 5' end of the donor DNA with 10 deoxyadenosines between them), (3) homology arms ("D20P16" indicates that the donor DNA has a distal homology arm 20 nucleotides long and a proximal homology arm 16 nucleotides long, and "D20P36" indicates that the donor DNA has a distal homology arm 20 nucleotides long and a proximal homology arm 36 nucleotides long), and (4) three SpCas9 (WT, HiFi, and eCas9). As expected, the number of off-target sites was reduced with high-fidelity Cas9 (see Figure 9B and Figure 9D), indicating that the internal anchor CRISPR system is compatible with high-fidelity Cas nucleases. Interestingly, the total number of GUIDE-seq reads was reduced when the internal anchor CRISPR system was used compared to the regular CRISPR system (see Figure 9E). Because the total number of GUIDE-seq reads is associated with NHEJ efficiency during integration of dsODNs with targeted double-strand breaks (DSBs), as part of the GUIDE-seq protocol, this result indicates that the internal anchor CRISPR system significantly suppressed the undesired NHEJ pathway.

[0123] Example 7: Long fragment deletion in HEK3 To delete long fragments in HEK3, dual modified sgRNAs (i.e., leg1 and leg2 in FIG. 10C) containing an internal anchor (sequence number: legRNA_Int_Anc) in the upper loop of the upper stem located at the first hairpin (the modified sgRNAs are also called "lead editing guide RNAs" and abbreviated as legRNA) were designed to target two different loci separated by 991-bp in HEK3 (FIG. 10A), SpCas9, and an anchored ssDNA (asODN) donor template composed of 36-base homology arms and legRNA anchor tails located upstream and downstream of the deletion site (FIG. 10B). Three asODN-legRNA approaches were designed and tested (FIG. 10C). Design A1: asODN1 (SEQ ID NO: HEK3_A1_asODN1), asODN2 (SEQ ID NO: HEK3_A1_asODN2), legRNA1 (SEQ ID NO: A1_HEK3_legRNA1) and legRNA2 (SEQ ID NO: A1_HEK3_legRNA2) target different strands; design A2: asODN3 (SEQ ID NO: HEK3_A2_asODN), and both legRNA3 (SEQ ID NO: A2_HEK3_legRNA1) and legRNA4 (SEQ ID NO: A2_HEK3_legRNA2) target the same top strand; design A2p: both legRNA5 (SEQ ID NO: A2p_HEK3_legRNA1) and legRNA6 (SEQ ID NO: A2p_HEK3_legRNA2) target the same bottom strand, and asODN4 (SEQ ID NO: HEK3_A2p_asODN) target another strand.

[0124] Experimental procedure Production of legRNA. legRNA was produced by in vitro transcription (IVT). Briefly, a DNA template for legRNA was constructed and amplified by PCR. The DNA was then transcribed into RNA using T7 polymerase, followed by DNAse I treatment to remove any remaining DNA template in the samples.

[0125] Transfection of Cas9-legRNA ribonucleoprotein (RNP). HEK293T cells were seeded in 24-well plates 1 day before transfection. First, legRNA was combined with cas9 protein to form a ribonucleoprotein complex (RNP). Then, asODN was added to form an RNP·asODN complex. The RNP·asODN complex was transfected into the cells using lipofectamine. Two to three days after transfection, DNA was extracted from the cells.

[0126] PCR and gel electrophoresis. The 1 kb deletion was visualized using PCR and gel electrophoresis. The PCR reaction used one forward and two reverse primers, allowing simultaneous amplification and visualization of the WT and deletion products on the gel.

[0127] Amplicon sequencing. Amplicon sequencing libraries were generated by two-step PCR using target site-specific primers and index primers. The libraries were sequenced by Illumine Nextseq500 or iSeq sequencing systems.

[0128] result The results of different asODN-legRNA designs are shown in Figure 11. Design A1 yielded deletion products with a deletion efficiency of approximately 30% of the WT product, i.e., approximately 23% (=30% / (100%+30%)) (Figures 11A and 11B), and the deletion was confirmed by sequencing (Figure 11C).

[0129] Example 8: Long fragment deletion in HPRT1 Similarly, we tested the deletion of a long fragment (1075-bp) in HPRT1 as in Example 1. We used a dual asODN-legRNA system similar to design A1 (asODN5: SEQ ID NO: HPRT_A1_asODN1; asODN6: SEQ ID NO: HPRT_A1_asODN2; legRNA7: SEQ ID NO: HPRT_legRNA1; and legRNA8: SEQ ID NO: HPRT_legRNA2), and the results are shown in Figure 12. Dual asODN-legRNA showed a deletion efficiency of up to about 86% (Figures 12A and 12B), but the deletion efficiency by gel intensity analysis may be overestimated because amplification favors short amplicons over long amplicons. We confirmed the deletion by sequencing (Figure 12C).

[0130] Example 9: Insertion of a large fragment in GAPDH To test the insertion of the 1734-nt large fragment into GAPDH, cas9 and legRNA (sequence number: GAPDH_legRNA) with circularized anchor ssODN (casODN), cas800HA (sequence number: casODN_800HA, internal anchor sequence: ACATTGTCTCACTT) were used (Figure 13A). casODN serves as an insertion template containing the anchor sequence, left homology arm, IRES, green fluorescent protein (ZsGreen) coding sequence, polyA, and right homology arm (Figure 13A). Transfection experimental conditions were similar to those in Example 1, except that two forms of cas9 were used: ribonucleotide (RNP) and plasmid (for comparison). For comparison, a normal double-stranded DNA template (dsONA), ds500HA (sequence number: dsODN_500HA), was also included. The results showed that RNP-casODN treatment resulted in a much stronger insertion product than the control without cas9 (Figure 13B). The low level of product in the no-cas9 control (lane 3 in Figure 13B) could be due to classical homologous recombination. When double-stranded templates were used, a 1734 base insertion was also seen, but substantial insertions were seen in those no-cas controls (lanes 4 and 5 in Figure 13B). Similar results were seen when cas9 was expressed by a plasmid (lanes 7-12 in Figure 13B). Edited cells with RNP-casODN treatment showed clear functional insertion of ZsGreen protein in treated cells, whereas no significant green signal was seen in the no-cas9 control cells (Figure 13C). Table 7. Nucleic acid sequences used in Examples 7-9 [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0131] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided as merely illustrative. The present disclosure is not intended to be limited by the specific examples provided herein. The present disclosure has been described with reference to the foregoing specification, but the description and drawings of the embodiments herein are not meant to be interpreted in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. Furthermore, it should be understood that all aspects of the present disclosure are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in carrying out the present invention. It is therefore contemplated that the present disclosure should cover any such alternatives, modifications, variations, or equivalents. The claims define the scope of the present disclosure, and it is intended that methods and configurations within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A system for altering a target sequence, comprising: a modified single guide RNA (sgRNA) and a donor DNA, wherein the modified sgRNA comprises a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA), wherein the modified sgRNA comprises one or more internal anchors at least 5 nucleotides away from both a 3' end and a 5' end of the modified sgRNA, and wherein the donor DNA comprises a first portion and a second portion, wherein the first portion comprises one or more binding segments capable of binding to one of the one or more internal anchors via a non-covalent bond, and wherein the second portion comprises a sequence of interest (SOI).

2. The system of claim 1 , wherein the non-covalent bond is a Watson-Crick interaction.

3. 3. The system of claim 1 or 2, wherein the modified sgRNA comprises a nexus, a first hairpin, and a single-stranded region between the tracrRNA and the crRNA.

4. The system of claim 3, wherein the modified sgRNA further comprises a bulge region.

5. 5. The system of claim 3 or 4, wherein the modified sgRNA further comprises a second hairpin.

6. 6. The system of claim 1, wherein the one internal anchor of the one or more internal anchors is located in a single-stranded region of the modified sgRNA.

7. The system of any one of claims 1 to 6, wherein the one internal anchor of the one or more internal anchors is located in the single-stranded region between the tracrRNA and the crRNA.

8. The system of any one of claims 1 to 7, wherein the one internal anchor of the one or more internal anchors is located in a single-stranded region within the first hairpin.

9. The system of any one of claims 1 to 8, wherein the one internal anchor of the one or more internal anchors is located in a single-stranded region between the nexus and the first hairpin.

10. 10. The system of any one of claims 1 to 9, wherein the modified sgRNA further comprises a second hairpin, and the one internal anchor of the one or more internal anchors is located in a single-stranded region within the second hairpin.

11. The system of any one of claims 1 to 10, wherein each of the one or more internal anchors or each of the one or more binding segments is between 3 nucleotides and 100 nucleotides in length.

12. 12. The system of claim 11, wherein each of the one or more internal anchors or each of the one or more binding segments is between 3 nucleotides and 20 nucleotides in length.

13. 13. The system of claim 12, wherein each of the one or more internal anchors or each of the one or more binding segments is about 5 nucleotides in length.

14. The system of any one of claims 1 to 13, wherein each of the one or more internal anchors comprises a sequence set forth in SEQ ID NOs: 1 to 472 of Table 1.

15. The system of any one of claims 1 to 14, wherein each of the one or more internal anchors comprises a sequence as set forth in SEQ ID NOs: 473 to 3056 of Table 2.

16. The system of any one of claims 1 to 14, wherein each of the one or more binding segments comprises a sequence as set forth in SEQ ID NOs: 3057 to 3528 of Table 3.

17. The system of any one of claims 1 to 14, wherein each of the one or more binding segments comprises a sequence as set forth in SEQ ID NOs: 3529 to 6112 of Table 4.

18. The system of any one of claims 1 to 17, wherein the one or more binding segments are linked by a linker.

19. The system of claim 18, wherein the linker is about 1 to 30 nucleotides in length.

20. The system of claim 19, wherein the linker is about 10 to 25 nucleotides in length.

21. The system according to any one of claims 18 to 20, wherein the linker is a polydeoxyadenosine sequence.

22. The system of any one of claims 1 to 21, wherein the SOI comprises the target sequence having one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions, or any combination thereof.

23. 23. The system of claim 22, wherein the one or more nucleotide insertions comprise between 1 and 100 nucleotides, between 101 and 1000 nucleotides, between 1001 and 10,000 nucleotides, or between 10,001 and 100,000 nucleotides.

24. 24. The system of claim 23, wherein the one or more nucleotide insertions comprise between 2 and 10 random nucleotides.

25. The system of claim 22, wherein the one or more nucleotide deletions comprise from 1 to 50 nucleotides.

26. The system of any one of claims 1 to 25, wherein the second portion of the donor DNA further comprises upstream and / or downstream homology arms.

27. The system of claim 26, wherein the upstream homology arm is between 5 and 1000 nucleotides in length.

28. The system of claim 26 or 27, wherein the downstream homology arm is about 10 to 1000 nucleotides in length.

29. The system of any one of claims 1 to 28, wherein the first portion of the donor DNA is located 5' to the second portion of the donor DNA.

30. The system of any one of claims 1 to 28, wherein the first portion of the donor DNA is located 3' to the second portion of the donor DNA.

31. The system of any one of claims 1 to 30, wherein the donor DNA is single-stranded.

32. The system of any one of claims 1 to 30, wherein the first portion of the donor DNA is single-stranded and the second portion of the donor DNA is fully double-stranded or partially double-stranded.

33. The system of any one of claims 1 to 32, wherein the donor DNA is closed at the 3' and / or 5' end.

34. The system of any one of claims 1 to 33, further comprising a CRISPR nuclease.

35. The system of claim 34, wherein the CRISPR nuclease is a DNA nuclease.

36. The system of claim 35, wherein the DNA nuclease is Cas9, Cas12, Cas14, or CasΦ.

37. 1. A system for cleaving a first locus on a first chromosome and a second locus on a second chromosome, comprising a donor DNA and two modified single guide RNAs (sgRNAs), each of the modified sgRNAs comprising a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA), each of the modified sgRNAs comprising one or more internal anchors at least 5 nucleotides away from both a 3' end and a 5' end of each of the modified sgRNAs, the donor DNA comprising a first portion and a second portion, the first portion comprising one or more binding segments capable of binding to one of the one or more internal anchors via a non-covalent bond, the second portion comprising a sequence of interest (SOI), and the donor DNA comprising an upstream homology arm and / or a downstream homology arm.

38. 38. The system of claim 37, wherein the first chromosome and the second chromosome are the same.

39. 39. The system of claim 37 or 38, wherein the first locus is located 5' to the second locus.

40. 38. The system of claim 37, wherein the first chromosome and the second chromosome are different.

41. 41. The system of any one of claims 37-40, wherein the first locus and the second locus are separated by at least 50, 100, 1,000, 10,000, or 100,000 nucleotides.

42. The system of any one of claims 37 to 41, wherein the upstream homology arm is adjacent to the 5' end of the first locus.

43. The system of any one of claims 37 to 42, wherein the downstream homology arm is adjacent to the 3' end of the second locus.

44. The system according to any one of claims 37 to 43, wherein the non-covalent bond is a Watson-Crick interaction.

45. 45. The system of any one of claims 37-44, wherein the modified sgRNA comprises a nexus, a first hairpin, and a single-stranded region between the tracrRNA and the crRNA.

46. 46. ​​The system of Claim 45, wherein the modified sgRNA further comprises a bulge region.

47. 47. The system of Claim 45 or 46, wherein the modified sgRNA further comprises a second hairpin.

48. 48. The system of any one of claims 37-47, wherein the one internal anchor of the one or more internal anchors is located in a single-stranded region of the modified sgRNA.

49. 49. The system of any one of claims 37 to 48, wherein the one internal anchor of the one or more internal anchors is located in the single-stranded region between the tracrRNA and the crRNA.

50. 50. The system of any one of claims 37 to 49, wherein the one internal anchor of the one or more internal anchors is located in a single-stranded region within the first hairpin.

51. 51. The system of any one of claims 37 to 50, wherein the one internal anchor of the one or more internal anchors is located in a single-stranded region between the nexus and the first hairpin.

52. 52. The system of any one of Claims 37-51, wherein the modified sgRNA further comprises a second hairpin, and the one internal anchor of the one or more internal anchors is located in a single-stranded region within the second hairpin.

53. 53. The system of any one of claims 37-52, wherein each of the one or more internal anchors or each of the one or more binding segments is between 3 nucleotides and 100 nucleotides in length.

54. 54. The system of claim 53, wherein each of the one or more internal anchors or each of the one or more binding segments is between 3 nucleotides and 20 nucleotides in length.

55. 55. The system of claim 54, wherein each of the one or more internal anchors or each of the one or more binding segments is about 5 nucleotides in length.

56. The system of any one of claims 37 to 55, wherein each of the one or more internal anchors comprises a sequence set forth in SEQ ID NOs: 1 to 472 of Table 1.

57. The system of any one of claims 37 to 55, wherein each of the one or more internal anchors comprises a sequence set forth in SEQ ID NOs: 473 to 3056 of Table 2.

58. The system of any one of claims 37 to 55, wherein each of the one or more binding segments comprises a sequence set forth in SEQ ID NOs: 3057 to 3528 of Table 3.

59. The system of any one of claims 37 to 55, wherein each of the one or more binding segments comprises a sequence set forth in SEQ ID NOs: 3529 to 6112 of Table 4.

60. The system of any one of claims 37 to 59, wherein the one or more binding segments are linked by a linker.

61. The system of claim 60, wherein the linker is about 1 to 30 nucleotides in length.

62. The system of claim 61, wherein the linker is about 10 to 25 nucleotides in length.

63. The system according to any one of claims 60 to 62, wherein the linker is a polydeoxyadenosine sequence.

64. 64. The system of any one of claims 37-63, wherein the SOI comprises a region between the first locus and the second locus having one or more nucleotide substitutions, one or more nucleotide insertions, one or more nucleotide deletions, or any combination thereof.

65. 23. The system of claim 22, wherein the one or more nucleotide insertions comprise between 1 and 100 nucleotides, between 101 and 1000 nucleotides, between 1001 and 10,000 nucleotides, or between 10,001 and 100,000 nucleotides.

66. 23. The system of claim 22, wherein the one or more nucleotide deletions comprise between 1 and 100 nucleotides, between 101 and 1000 nucleotides, between 1001 and 10,000 nucleotides, or between 10,001 and 100,000 nucleotides.

67. The system of any one of claims 37 to 66, wherein the upstream homology arm is 5 to 1000 nucleotides in length.

68. 68. The system of any one of claims 37 to 67, wherein the downstream homology arm is about 10 to 1000 nucleotides in length.

69. The system of any one of claims 37 to 68, wherein the first portion of the donor DNA is located 5' to the second portion of the donor DNA.

70. The system of any one of claims 37 to 68, wherein the first portion of the donor DNA is located 3' to the second portion of the donor DNA.

71. The system of any one of claims 37 to 70, wherein the donor DNA is single-stranded.

72. 71. The system of any one of claims 37 to 70, wherein the first portion of the donor DNA is single-stranded and the second portion of the donor DNA is fully double-stranded or partially double-stranded.

73. The system of any one of claims 37 to 72, wherein the donor DNA is closed at the 3' and / or 5' end.

74. The system of any one of claims 37 to 73, wherein the system further comprises a CRISPR nuclease.

75. 75. The system of claim 74, wherein the CRISPR nuclease is a DNA nuclease.

76. The system of claim 75, wherein the DNA nuclease is Cas9, Cas12, Cas14, or CasΦ.

77. 77. A method for modifying a cell, comprising delivering a system according to any one of claims 1 to 76 into said cell.

78. The transport, a. incubating the CRISPR nuclease and the modified sgRNA to form a ribonucleoprotein (RNP) complex; b. applying the donor DNA to the RNP complex; c. delivering the RNP complex-donor DNA from (b) to the cell.

79. 79. The method of Claim 78, wherein in step (a), the ratio of the CRISPR nuclease to the modified sgRNA is from about 1:0.5 to about 1:

10.

80. 79. The method of claim 78, wherein in step (a), the ratio of the CRISPR nuclease to the modified sgRNA is about 1:1 to 1:

2.

81. The transport, a. providing one or more vectors comprising a nucleotide sequence encoding the CRISPR nuclease and a nucleotide sequence encoding the modified gRNA; b. delivering the one or more vectors in (a) to the cell; c. delivering the donor DNA to the cell.

82. 82. The method of claim 81, wherein step (c) is carried out about 6 to 48 hours after step (b) is completed.

83. 83. The method of any one of claims 77 to 82, wherein the delivery is achieved by a viral vector, a liposome, a lipid nanoparticle, or electroporation.

84. The method of any one of claims 77 to 83, wherein the cell is an immune cell.

85. 85. The method of claim 84, wherein the immune cell is a T cell, a B cell, a NK cell, or a hematopoietic stem cell.

86. The method of any one of claims 77 to 85, wherein the method is performed ex vivo or in vivo.

87. 87. The method of any one of claims 77-86, wherein the percentage of desired editing is at least 10%, at least 50%, at least 100%, or at least 200% higher than a similar system that does not include the donor DNA comprising the first portion that binds to the modified sgRNA and / or does not include the modified sgRNA with the one or more internal anchors.

88. 88. The method of any one of claims 77-87, wherein the method has an off-target rate that is at least 10%, at least 50%, or at least 100% lower than a similar system that does not include the donor DNA comprising the first portion that binds to the modified sgRNA and / or does not include the modified sgRNA with the one or more internal anchors.

89. 89. The method of any one of claims 77-88, wherein the method has a translocation rate, large insertion rate, or large deletion rate that is at least 10%, at least 50%, or at least 100% lower than a similar system that does not include the donor DNA comprising the first portion that binds to the modified sgRNA and / or does not include the modified sgRNA with the one or more internal anchors.

90. 80. A method for treating a genetic disease, comprising administering to a subject an effective amount of the system of claims 1-76.

91. 91. The method of claim 90, wherein the SOI comprises a sequence that reverses or alleviates the genetic disease.

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