Enhancing safety and accuracy of CRISPR-CAS-induced gene editing through DNA polymerase variants using CAS-PLUS variants

Abstract

The present invention provides compositions and methods comprising an engineered DNA polymerase for use in combination with a Cas9 protein. The combination exhibits improved in-target chromosomal alterations, increases the proportion of precise 1 to 3 base pair insertions at the target site, and reduces translocation caused by previously available systems.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 335,625 filed on April 27, 2022 and U.S. Provisional Patent Application No. 63 / 433,353 filed on December 16, 2022, the entire disclosure of each application is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted in .xml format and is incorporated herein by reference in its entirety. The name of the .xml file is "CRISPR-Cas INDUCED GENE EDITING.xml", created on April 26, 2023, and is 107,424 bytes in size. Background Art

[0005] The engineered CRISPR / Cas9 system is a powerful tool for sequence-specific gene editing (1-4) However, it may also produce undesirably large deletions (5,6) , Chromosomal translocation (7) , Chromosome fragmentation (8) and other complex chromosomal rearrangements and off-target effects. Although many strategies have been developed to minimize CRISPR / Cas9-mediated off-target effects (9) Cas9 cleaves target DNA to produce blunt ends or staggered ends with 5' overhangs. (10) Repair of these ends is usually performed by classical nonhomologous end joining (c-NHEJ) or microhomology-mediated end joining (MMEJ). (11) The choice of repair pathway determines the outcome of CRISPR / Cas9 editing. MMEJ repair often results in deletions, especially large deletions. (12,13) Systematic analysis of Cas9 target sites shows that insertions from the c-NHEJ pathway are precise and predictable (14-16) The frequency and pattern of insertions are highly dependent on the local sequence surrounding the Cas9 cleavage site. (17) . But there are limited ways to enhance these results. Therefore, there remains a constant need for improved safety and precision of Cas enzyme-based DNA editing. The present disclosure is relevant to this need. Summary of the invention

[0006] The present disclosure provides compositions and methods for precise genome editing. The composition includes a DNA polymerase, and representative examples thereof are further described below. In an embodiment, the present disclosure provides a fusion protein, the fusion protein comprising: a DNA polymerase fragment, the fragment may include amino acid sequence changes relative to a reference DNA polymerase sequence (i.e., a wild-type DNA polymerase sequence), representative amino acid changes will be further described herein, and a fragment of an MS2 bacteriophage coat protein. A separate DNA polymerase or the fusion protein is operated with Cas and one or more guide RNAs to produce one or more insertions / deletions. Cas may also include amino acid sequence changes relative to a reference sequence (i.e., a wild-type Cas sequence), and representative amino acid changes will be further described herein.

[0007] In an embodiment, non-homologous end joining (NHEJ) is used to generate insertions / deletions, which are at least partially promoted by the DNA polymerase, which is a component of the genome editing system encompassed by the present disclosure. The present disclosure provides for generating insertions / deletions in a manner without a DNA repair template. The protein functions as a component of the CRISPR system in the nucleus. Therefore, any protein described herein may include at least one nuclear localization signal. When the fusion protein is used, it may also include one or more joints, which, for example, separate the DNA polymerase from MS2, and / or separate the fragment of the fusion protein from the nuclear localization signal. In an embodiment, the fusion protein comprises a self-cleaving peptide sequence, which may, for example, promote ribosome jumping during translation. Therefore, the fusion protein may be encoded by an mRNA encoding additional amino acids at the N-terminus or C-terminus of the fusion protein, and these amino acids are not translated as part of a continuous polypeptide comprising a DNA polymerase and an MS2 protein fragment by the operation of the self-cleaving peptide sequence.

[0008] In one aspect, the present disclosure includes a complex comprising a Cas enzyme, a guide RNA, a protein, the guide RNA optionally comprising an MS2 phage coat protein binding site, the protein comprising a DNA polymerase and optionally also comprising an MS2 binding protein. In a non-limiting embodiment, when a DNA polymerase is used together with an MS2 protein component, the guide RNA comprises an MS2 protein binding sequence. Also included are cells comprising the DNA polymerase or a fusion protein comprising a DNA polymerase and a guide RNA. Also provided are pharmaceutical compositions comprising the protein. Such compositions may also comprise guide RNA and a Cas enzyme. Also included are cells comprising the protein and the complex. The present disclosure also provides expression vectors and cDNA encoding the protein, as well as kits comprising them and / or other components.

[0009] In embodiments, the disclosure provides for reducing translocation events.For example, in the case where Cas9 or other site-specific nucleases (except the CasPlus system) target more than one chromosome position, cutting at more than one position on one or more chromosomes will produce the risk of proven translocation events. The disclosure proves that such translocation events can be reduced by using the CasPlus system. Therefore, the CasPlus system can be used, for example, to destroy one or more genes with different targeting guide RNAs and produce insertions / deletions at more than one position, while reducing the possibility of translocation relative to other DNA editing enzymes. In embodiments, compared with previous methods, the reduction of translocation events is achieved in any eukaryotic cell type, and the cells include but are not limited to lymphocytes and leukocytes, such as T cells, and the T cells include but are not necessarily limited to T cells expressing chimeric antigen receptors (CAR) or other types of genetically modified T cells that can be modified using any other guide-guided nucleases.

[0010] In another aspect, the present disclosure provides a method for producing insertions / deletions at a selected chromosomal site in a cell. The method comprises introducing the protein, the Cas enzyme, and a guide RNA optionally comprising an MS2 protein binding site into a cell, wherein the guide RNA guides the Cas enzyme, the DNA polymerase, and optionally the MS2 binding protein to the selected chromosomal site, thereby producing insertions / deletions. In an embodiment, the insertion / deletion corrects a mutation in an open reading frame encoded by the selected chromosomal site or converts the sequence to an open reading frame. In an embodiment, the selected chromosomal site includes a mutation in a gene associated with a monogenic disease. In a non-limiting embodiment, the monogenic disease is muscular dystrophy, wherein the selected chromosomal site includes a gene for a dystrophin protein comprising a mutation. In this regard, Duchenne muscular dystrophy (DMD) is a debilitating neuromuscular disease that causes degeneration of heart and skeletal muscle. (18) , and is caused by inactivating mutations in the X-linked dystrophin gene (DMD) (19) Dilated cardiomyopathy (DCM) is a common and fatal feature of DMD (20) We have previously used CRISPR-Cas9 to correct DMD mutations in cultured human cells and mdx mice. (21-23); However, the undesirable DNA damage at the edited DMD site has not been evaluated, which is a safety issue in human therapy. Therefore, in embodiments, compared with previous methods, insertion / deletion, for example, corrects the gene encoding the mutated dystrophin with a lower off-target modification frequency. In some instances, insertion / deletion includes one or two base pair insertions. In embodiments, the monogenic disease is cystic fibrosis, wherein the selected chromosomal site includes a gene comprising a mutant protein gene associated with cystic fibrosis. In one embodiment, the system corrects F508del in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A-1D .Identification of T4 and RB69 DNA polymerases as proteins that facilitate CasPlus editing. Figure 1A Schematic diagram showing two functions of the wild-type T4 DNA polymerase-mediated CasPlus system in cells: enhancing 1-bp insertions by promoting staggered end filling (top DNA repair pathway) and suppressing MMEJ-dependent deletions by disrupting annealing of MH (bottom DNA repair pathway). Figure 1B The workflow shows the DNA polymerase selection process in tdTomato reporter cells. Briefly, vectors expressing Cas9, GFP, or tdTomato-sgRNA alone or in combination with different DNA polymerases were transfected into tdTomato reporter cells. Transfected cells were sorted to express only GFP (tdTomato - / GFP + ) or expressing both tdTomato and GFP (tdTomato + / GFP + ) for DNA isolation and high-throughput sequencing. Figure 1C Frequency of Cas9-induced indels when overexpressing Cas9 alone (referred to as CTR) or in combination with T4, RB69, and T7 DNA polymerases in tdTomato reporter cells. + / GFP + and tdTomato - / GFP + Cells were sorted as described above. The upper and lower dashed lines show the deletion and 2-bp insertion frequencies (CTR) in cells treated with Cas9 alone, respectively. Figure 1DTemplate-dependent insertions of one or two base pairs in all treatment groups. Templated 1-bp insertions indicate the insertion of one nucleotide identical to the nucleotide at position -4, whereas templated 2-bp insertions indicate the insertion of two nucleotides identical to the nucleotides at positions -5 and -4 (if the NGGPAM sequence is counted as positions 0-2). Figure 1E Western blot analysis performed in tdTomato reporter cells overexpressing T4, RB69, and T7 DNA polymerases. Arrows point to the correct size band for each DNA polymerase.

[0012] Figures 2A-2H .T4 DNA polymerase mutant D219A (T4-D219A) improves the efficiency of T4 DNA polymerase-mediated CasPlus editing. Figure 2A .Schematic diagram showing that engineered T4 DNA polymerase mutants can promote the filling process of Cas9-induced DSB ends with 1-bp overhangs and 1-bp insertion. Figure 2B Schematic diagram showing the positions of all tested T4 DNA polymerase mutants and the corresponding DNA mutation frequencies induced by the mutants relative to T4-WT DNA polymerase. The mutation frequencies were calculated according to published literature (24-26). Figure 2C .Frequency of Cas9-induced insertions / deletions at TS11 in cells co-overexpressing CTR or Cas9 and T4 DNA polymerase mutants. The sequence of TS11 is shown in Table 1. The upper and lower dashed lines show the frequencies of deletions and 1-bp insertions in cells overexpressing Cas9-WT and T4-WT, respectively. Arrows point to bars representing 1-bp insertions (left) and deletions (right) in cells overexpressing Cas9-WT and T4-D219A. Figure 2D -F. TS2, TS10 and TS12 in CTR, T4-WT or T4-D219A overexpressing cells ( Figure 2D ), TS17 and TS18( Figure 2E ) or TS26( Figure 2F ) The frequency of Cas9-induced indels at . Relative to T4-WT, the T4-D219A mutant improved the insertion frequency at the expense of deletions that occurred at all genomic sites shown. The target site sequences are shown in Table 1. Figure 2G .Schematic diagram showing the ability of T4 DNA polymerase to fill in the 5-8 bp overhangs generated by Cas12a. Figure 2H .Indel frequencies induced by Cas12a in cells transfected with Cas12a alone or co-transfected with Cas12a and T4-WT or T4-D219A. The sequence of guide RNA Lb1 is shown in Table 1.

[0013] Figure 3A-3B.RB69 DNA polymerase mutant D222A (RB69-D222A) improves the efficiency of RB69 DNA polymerase-mediated CasPlus editing. Figure 3A .tdTomato isolated from tdTomato reporter cells co-transfected with Cas9-WT and RB69-WT or RB69-D222A + / GFP + Cells and tdTomato - / GPF + Frequency of Cas9-induced indels in cells. Figure 3B Frequency of Cas9-induced indels at TS2, TS11, and TS12 in cells co-transfected with Cas9-WT and RB69-WT or RB69-D222A. The RB69-D222A mutant improved the insertion frequency at these genomic sites.

[0014] Figures 4A-4F .At Cas9 target sites where Cas9-WT and T4-WT primarily produced deletions, the combination of Cas9 variants with T4 DNA polymerase enhanced 1-bp insertions. Figure 4A Schematic showing sites where Cas9-WT induces blunt-ended DSBs and deletions, and where some engineered Cas9 variants can promote the generation of 1-bp overhangs at these sites, so that addition of T4 DNA polymerase can generate 1-bp insertions. Figure 4B .Schematic diagram showing the mutation sites of the tested Cas9 variants. All mutations were within the linkage II (L-II) region. Figure 4C .Frequency of Cas9-induced indels at TS11 in cells transfected with Cas9-WT or Cas9 variants. The upper and lower dashed lines show the frequency of deletions and 1-bp insertions in cells overexpressing Cas9-WT, respectively. Arrows point to bars representing 1-bp insertions or deletions in cells overexpressing Cas9 variants F916P, F916del, F919P, or Q920P. Figure 4D Frequency of Cas9-induced indels at TS11 in cells co-transfected with T4-WT and Cas9-WT or Cas9 variants. Figure 4E-4F Frequency of Cas9-induced indels at TS19 or TS22 (E), TS24, TS25, and TS26 (F) in cells transfected with Cas9-WT, Cas9 variants F916P or F916del alone or in combination with T4-WT or T4-D219A. Arrows point to bars representing 1-bp indels in cells that exhibited increased 1-bp indels at the expense of indels compared to cells with Cas9-WT overexpression alone.

[0015] Figures 5A-5E The combination of Cas9 variants and T4 DNA polymerase enhanced the production of longer inserts (2 to 4 bp). Figure 5A Schematic showing that at sites where Cas9-WT generates DSB ends with 1-bp overhangs, leading to edits with 1-bp insertions, engineered Cas9 variants can promote the generation of 2-bp overhangs at these sites, leading to 2-bp insertions in the presence of T4 DNA polymerase. Figure 5B GFP isolated from tdTomato reporter cells transfected with Cas9 or Cas9 variants + Frequency of Cas9-induced indels in the population. Figure 5C GFP isolated from tdTomato reporter cells co-transfected with T4-WT and Cas9-WT or Cas9 variants + Frequency of Cas9-induced indels in the population. Arrows point to bars representing 3-bp insertions. Figure 5D . Frequency of Cas9-induced indels at TS5, TS17, and TS18 in cells transfected with Cas9-WT, Cas9 variant F916P, or Cas9 variant F916del alone or in combination with T4-WT or T4-D219A. Arrows point to bars representing significantly increased longer indels in cells co-transfected with T4 DNA polymerase and Cas9 variants F916P or F916del compared to cells co-transfected with T4-WT and Cas9-WT. Figure 5E .Design of different versions of T4 DNA polymerase-mediated CasPlus systems. CasPlus-V1 is a combination of Cas9-WT and T4-WT. CasPlus-V2 labeled a combination of Cas9-WT and T4-D219A. CasPlus-V3 and V4 use a combination of Cas9 variants and T4-WT or T4-D219A, respectively. CasPlus-V3 and V4 are further divided into subcategories based on the Cas9 variants used. Cas9 variants F916P, F916del, R920P, and Q920P are named V3.1, V3.2, V3.3, and V3.4 in CasPlus-V3, respectively; or V4.1, V4.2, V4.3, and V4.4 in CasPlus-V4, respectively. All T4 DNA polymerases are MS2-targeted.

[0016] Figures 6A-6G The CasPlus system effectively suppresses large deletions. Fig. 6A . Schematic showing that CasPlus suppresses large deletions by inhibiting long-distance end resection. Figure 6B . Schematic diagram showing the location of primer sets used to amplify the distal or proximal regions of TS10. Figure 6C . Induced pluripotent stem cells (iPSCs) with DMD exon 52 deletion were transfected with Cas9, CasPlus-V1 or CasPlus-V2 to target DMD exon 51. Sorting and isolation of GFP + Cells were subjected to PCR amplification. The PCR gel image is shown on the left, and the Sanger sequencing results of the lower band are shown on the right. Figure 6C The sequence in is 5'-GGTGGGTGACCTGGGAATTGATTATT-3' (SEQ ID NO: 1). Fig.6D . Schematic diagram showing the location of primer sets used to amplify the distal or proximal regions of TS9. Fig. 6E Transfection of induced pluripotent stem cells (iPSCs) with DMD exon 52 deletion using Cas9, CasPlus-V1, or CasPlus-V2 to target DMD exon 53. Sorting and isolation of GFP + Cells were subjected to PCR amplification. The PCR gel image is shown on the left, and the Sanger sequencing results of the lower band are shown on the right. Figure 6F-6G In untreated, Cas9-, CasPlus-V1-, and CasPlus-V2-edited iPSCs with DMD exon 52 deletion, DMD exon 51 ( Fig. 6F ) or 53( Figure 6G ) at PacBio read depth. Figure 6C The sequence in is: 5'-GGTGGGTGACCTGGGAATTGATTATT-3' (SEQ ID NO: 1). Fig. 6E The sequence in is: 5'-TATTTTAATATTTGTCAGTGGGATGA-3' (SEQ ID NO: 2).

[0017] Figures 7A-7F .Correction of DMD exon 52 deletion in iPSCs enhanced by CasPlus editing. Fig. 7A Deletion of exon 52 in DMD results in the generation of a premature stop codon in exon 53, thereby abolishing dystrophin expression. Two strategies can be used to restore dystrophin expression via 1-bp insertions edited by CasPlus. Figure 7B All available guide RNAs containing NGG as the PAM sequence are shown at the 3' end of DMD exon 51 (TS10 and TS27) and at the 5' end of exon 53 (TS9, TS28, TS29, TS30, and TS31). Figure 7CFrequency of 1-bp insertions, other remodeled indels (3n+1, n≠0), or other indels (3n and 3n+2) induced by Cas9 in iPSCs transfected with Cas9, CasPlus-V1, or CasPlus-V2. Fig.7D Frequency of mRNA alleles with 1-bp insertions, other remodeled indels, or other indels in cardiomyocytes differentiated from iPSCs transfected with Cas9, CasPlus-V1, or CasPlus-V2. SC. Single clones with 1-bp insertions selected from the TS10 or TS9 edited cell pool were used here as positive controls. Fig. 7E .RT-PCR analysis of cardiomyocytes differentiated from iPSCs transfected with Cas9, CasPlus-V1 or CasPlus-V2. Cells transfected with Cas9 induced skipping of the entire exon 51 or exon 53 (the lower band with arrows). The Sanger sequencing results of the lower band are shown on the right. Figure 7F . Western blot analysis of cardiomyocytes differentiated from iPSCs transfected with Cas9, CasPlus-V1, or CasPlus-V2. Figure 7B The sequence of exon 51 is: top: 5'-TGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAGGTATGA -3' (SEQ ID NO: 3); bottom: 5'-TCATACCTTCTGCTTGATGATCATCTCGTTGATATCCTCAAGGTCA-3' (SEQ ID NO: 4). The sequence of exon 53 is: top: 5'-aGTTGAAAGAATTCAGAATCAGTGGGATGAAGTACAAGAACACCTTCAGAACCGGAGGCAACAGTT; and GA-3' (SEQ ID NO: 5) and bottom: 5'-TCAACTGTTGCCTCCGGTTCTGAAGGTGTTCTTGTACTTCATCCCACTGATTCTGAATTCTTTCAACT-3' (SEQ ID NO: 6). Fig. 7E The sequence of exon 50-exon is: 5′-CACTATTGGAGCCTTTGAAAGAATTCAG-3′ (SEQ ID NO: 7); Fig. 7E The sequence of exon 51 to exon 54 is: 5′-TCATCAAGCAGAAGCAGTTGGCCAAAGA-3′ (SEQ ID NO: 8).

[0018] Figures 8A-8JExogenous template-independent correction of the CFTR F508del mutation by sequential CasPlus editing. Fig. 8A Schematic diagram showing the target exons from a wild-type individual (top sequence) and a CFTRF508del patient (bottom sequence) with the CFTR F508del mutation. The nucleotides deleted in the CFTR-F508del patient are marked with a red dashed line. Figure 8B . Schematic diagram showing the sequences of guide RNA, PAM, and single-stranded oligodeoxynucleotide (ssODN) template used to generate the CFTR-F508del knock-in HEK293T cell line. Figure 8C Schematic diagram showing four potential strategies for correction of the CFTR mutation F508del by CasPlus. One-step insertion of 3 bp generated an allele with a missense mutation. Two- or three-step incorporation of 3 bp by sequential CasPlus editing corrected the mutant allele. Fig.8D Guide RNA and PAM sequences for sequential correction of CFTR-F508del mutation. TS32 was designed to target the CFTR-F508del mutant allele, TS33 was used to target the intermediate mutant product with thymine insertion, and TS34 and TS36 were used to target the intermediate mutant products with AT or TT insertion, respectively. Fig. 8E .Indel spectra and frequencies induced by Cas9 editing (including Cas9-NG-WT and Cas9-NG-F916del) and CasPlus editing using guide RNA TS32 in CFTR-F508del HEK293T cells. CasPlus editing mainly promotes the generation of 1-bp and 2-bp insertions. Cas9-NG is a Cas9 variant that recognizes the NGN PAM sequence. Figure 8F-8G .Indel spectra and frequencies induced by two-step sequential CasPlus editing. Figure 8F The editing results of the combination of CasPlus-V1 and CasPlus-V2 with guide RNAs TS32 and TS33 or guide RNAs TS32 and 34 are shown. Figure 8G Editing results of CasPlus-V3.1 and CasPlus-V4.1 in combination with guide RNAs TS32 and 33 or TS32 and 34 are shown. Figure 8H .Indel spectra and frequencies induced by sequential CasPlus editing with guide RNAs TS32, TS33, and TS34 or combinations of TS32, TS33, and TS35. Figure 8I . Fig.8F and Figure 8G Pattern of 3-bp insertions detected in . Figure 8J . Figure 8H Pattern of 3-bp insertions detected in Fig. 8A , the sequence of WT is: 5'-GCACCATTAAAGAAAATATCATCTTTGG-3' (SEQ ID NO: 9); the sequence of F508del is: 5'-GCACCATTAAAGAAAATATCATTGG-3' (SEQ ID NO: 10). Figure 8B , the sequence of CFTR-WT is: 5'-CACCATTAAAGAAAATATCATCTTTGG-3' (SEQ ID NO: 11); the sequence of ssODN is: 5'-CCAATGATATTTTCTTTAATGGTGC-3' (SEQ ID NO: 12). Figure 8C , the WT sequence is: AATATCATCTTTGGTGTT (SEQ ID NO: 13); the missense sequence is: AATATCATCATTGGTGTT (SEQ ID NO: 14); the corrected sequences are AATATCATATTTGGTGTT (SEQ ID NO: 15) and AATATCATTTTTGGTGTT (SEQ ID NO: 16). Fig.8D , the sequence of CFTR-F508del is: top: 5'-ATTAAAGAAAATATCATTGGTGTTTCCTATGATGA-3' (SEQ ID NO: 17); bottom: 5'-TCATCATAGGAAACACCAATGATATTTTCTTTAAT-3' (SEQ ID NO: 18); the sequence of CFTR-F508del+T is: top: 5'-ATTAAAGAAAATATCATTTGGTGTTTCCTATGATGA-3' (SEQ ID NO: 19); bottom: 5'-TCATCATAGGAAACACCAAATGATATTTTCTTTAAT-3' (SEQ ID NO: 20); the sequence of CFTR-F508del+AT is: top: 5'-ATTAAAGAAAATATCATATTGGTGTTTCCTATGATGA-3' (SEQ ID NO: 21); bottom: 5'-TCATCATAGGAAACACCAATATGATATTTTCTTTAAT-3' (SEQ ID NO: 22); the sequence of CFTR-F508del+TT is: top: 5'-ATTAAAGAAAATATCATTTTGGTGTTTCCTATGATGA-3' (SEQ ID NO: 23); bottom: 5'-TCATCATAGGAAACACCAAAATGATATTTTCTTTAAT-3' (SEQ ID NO: 24).

[0019] Figures 9A-9H .CasPlus editing suppresses balanced on-target chromosomal translocation between two chromosomes. Fig. 9A .CasPlus editing inhibits Cas9-mediated chromosomal translocation. Fig. 9B . Schematic diagram illustrating the generation of chromosomes with ROS1-CD74 or CD74-ROS1 fusions. Fig. 9C Representative gel images showing ROS1-CD74 and CD74-ROS1 translocations in HEK293T cells during editing with Cas9, CasPlus-V1, or CasPlus-V2. HEK293T cells were transfected with vectors expressing Cas9 (with T2A-GFP) and guide RNAs targeting genes ROS1 and CD74 or with vectors expressing T4-WT or T4-D219A alone. Transfected cells were sorted for GFP 72 hours after transfection. + The population was immediately subjected to DNA isolation. DMD was a control used for intensity normalization. Fig.9D Normalized quantification of data in C. The band intensity obtained from Cas9-edited cells was set to 1. Values ​​and error bars reflect mean ± SEM of n = 3 replicates. Fig.9E Indel frequencies at individual loci of ROS1 and CD74 in HEK293T cells during Cas9, CasPlus-V1, or CasPlus-V2 editing. Values ​​and error bars reflect mean ± SEM of n = 3 replicates. Fig.9F Representative gel images showing ROS1-CD74 and CD74-ROS1 translocations in iPSC cells. Induced pluripotent stem cells were transfected with a vector expressing Cas9 (with T2A-GFP) and guide RNAs targeting genes ROS1 and CD74 and with vectors expressing T4-WT or T4-D219A alone. Transfected cells were sorted for GFP 72 hours after transfection. + The colonies were immediately subjected to DNA isolation. Figure 9G . Fig.9F Normalized quantification of the data. Figure 9H Indel frequencies of individual ROS1 and CD74 loci in iPSCs. Fig. 9C , the sequence of Chr6-Chr5:ROS1-CD74 is: 5'-GAAGCAAAGGG-3' (SEQ ID NO: 25); the sequence of Chr5-Chr6:CD74-ROS1 is: 5'-GAAGTACAGGCT-3' (SEQ ID NO: 26).

[0020] Figures 10A-10D .CasPlus editing suppresses balanced on-target chromosomal translocations between multiple chromosomes. Fig. 10A . Schematic diagram illustrating a balanced translocation between the genes PDCD1, TRBC1 / 2, and TRAC. Fig. 10B Representative gel images showing balanced translocations detected in HEK293T cells during editing with Cas9, CasPlus-V1, or CasPlus-V2. HEK293T cells were transfected with vectors expressing Cas9 (with T2A-GFP) and guide RNAs targeting genes PDCD1, TRBC1 / 2, and TRAC and with vectors expressing T4-WT or T4-D219A alone. Transfected cells were sorted for GFP 72 hours after transfection. + The colony was immediately subjected to DNA isolation. The band with the expected size (red arrow) was purified, TA cloned and sequenced. No balanced translocation of Chr14:Chr2, TRAC-PDCD1 was detected by PCR. Fig. 10C . Fig. 10B Normalized quantification of data in . Values ​​and error bars reflect mean ± SEM of n = 2 replicates. Fig. 10D Frequency of out-of-frame and in-frame indels at four individual loci in HEK293T cells during editing with Cas9, CasPlus-V1, or CasPlus-V2. Values ​​and error bars reflect mean ± SEM of n = 2 replicates. Fig. 10B , the sequence of Chr2-Chr7:PDCD1-TRBC1 is: 5'-CCCAGACCCAGG-3' (SEQ ID NO: 27); the sequence of Chr2-Chr7:PDCD1-TRBC2 is: 5'-AGCCCACCCAGG-3' (SEQ ID NO: 28); the sequence of Chr2-Chr14:PDCD1-TRAC is: 5'-CCCAGATCTATG-3' (SEQ ID NO: 29); the sequence of Chr7-Chr2:TRBC1 / 2-PDCD1 is: 5'-AGTGGACGACTG-3' (SEQ ID NO: 30); the sequence of Chr7-Chr14:TRBC1 / 2-TRAC is: 5'-AGTGGATCTATG-3' (SEQ ID NO: 31); the sequence of Chr14-Chr7:TRAC-TRBC1 is: 5'-TGAGGTCCCAGG-3' (SEQ ID NO:32); the sequence of Chr14-Chr7:TRAC-TRBC2 is: 5'-TGAGGTCCCAGG-3' (SEQ ID NO:33).

[0021] Figures 11A-11C .CasPlus editing suppresses unbalanced on-target chromosomal translocations between multiple chromosomes. Fig.11A. Schematic diagram illustrating six interchromosomal unbalanced translocations between the genes PDCD1, TRBC1 / 2, and TRAC. Fig. 11B Gel images showing unbalanced translocations induced by Cas9, CasPlus-V1, or CasPlus-V2 and guide RNAs targeting PDCD1, TRBC1 / 2, and TRAC. Bands with expected sizes (red arrows) were purified, TA cloned, and sequenced. Fig. 11C . Fig. 11B Quantification of data in . Values ​​and error bars reflect mean ± SEM of n = 2 replicates. Fig. 11B , the sequence of Chr2-Chr7 (no centromere) (PDCD1-TRBC1) is: 5'-GCGCCCAGGATA-3' (SEQ ID NO: 34); the sequence of Chr2-Chr7 (no centromere) (PDCD1-TRBC2) is: 5'-CCAGTCCCCAGG-3' (SEQ ID NO: 35); the sequence of Chr2-Chr14 (no centromere) (PDCD1-TRAC) is: 5'-CCAGTCTATGGA-3' (SEQ ID NO: 36); the sequence of Chr2-Chr7 (dicentric) (TRBC1 / 2-PDCD1) is: 5'-AGTGGATCTGGG-3' (SEQ ID NO: 37); the sequence of Chr2-Chr14 (dicentric) (TRAC-PDCD1) is: 5'-TGAGGTTCTGGG-3' (SEQ ID NO:38); the sequence of Chr7-Ch14 (no centromere) (TRBC1-TRAC) is: 5'-CCTGGGGACTTC-3' (SEQ ID NO:39); the sequence of Chr7-Chr14 (no centromere) (TRBC2-TRAC) is: 5'-CCTGGGCTATGG-3' (SEQ ID NO:40); the sequence of Chr7-Chr14 (double centromere) (TRBC1 / 2-TRAC) is: 5'-AGTGGAACCTCA-3' (SEQ ID NO:41).

[0022] Fig.12 Features of CasPlus editing. CasPlus editing utilizes T4 DNA polymerase to fill in overhangs generated by Cas9, thereby favoring insertions over small or large deletions. CasPlus editing can also inhibit chromosomal translocations that can occur between on-target and off-target sites during Cas9-mediated single-site editing or between different on-target genes during multiplex gene editing. DETAILED DESCRIPTION

[0023] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0024] Unless otherwise indicated, every maximum numerical limitation given in this specification includes every lower numerical limitation, as if these lower numerical limitations were expressly written herein. Every minimum numerical limitation given in this specification will include every higher numerical limitation, as if these higher numerical limitations were expressly written herein. Every numerical range given in this specification will include every narrower numerical range that falls within such broader numerical range, as if these narrower numerical ranges were expressly written herein.

[0025] The present disclosure includes all polynucleotides and all amino acid sequences indicated by database entries herein. Such sequences are incorporated herein because they are present in the database on the filing date of the present application or patent. Complementary and antiparallel polynucleotide sequences are included. The present disclosure encompasses every DNA and RNA sequence encoding the polypeptide disclosed herein. Also included are the amino acids of all protein sequences and all polynucleotide sequences encoding them, including but not limited to the sequences included by sequence alignment. Includes sequences that are 80.00%-99.99% identical to any sequence (amino acid and nucleotide sequence) disclosed herein. The nucleotide and amino acid sequences described herein include all continuous fragments of the nucleotide sequences that are at least 10 nucleotides or 10 amino acids in length.

[0026] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges and other values ​​may be expressed herein as from "about" or "approximately" one particular value, and / or to "about" or "approximately" another particular value. When values ​​are expressed as approximations by use of the antecedent "about" or "approximately," it should be understood that the particular value forms another embodiment. The terms "about" and "approximately" in relation to numerical values ​​encompass variations of + / -10% to + / -1%.

[0027] The present disclosure includes all steps and reagents described herein and shown in the drawings, such as proteins and nucleic acids, and all combinations of steps and reagents. The steps can be performed in the described manner, including but not limited to sequential execution.

[0028] In certain embodiments, the amino acid sequences described herein may refer to sequences lacking the original Met. For example, for the T4 DNA polymerase amino acid sequence, the mutation described at position 219 may appear at position 218 in the amino acid sequence due to the expression vector cloning process.

[0029] In an embodiment, the present disclosure provides a variant of the T4 DNA polymerase / Cas9 system referred to as "CasPlus". The variant of the CasPlus system is referred to herein as CasPlus-V1, which includes a combination of Cas9-WT and T4-WT in addition to other components described. Cas9 and the variants are amino acid sequences ("SpCas9") of Cas9 produced by Streptococcus pyogenes. CasPlus-V2 includes a combination of Cas9-WT and T4-D219A in addition to other components described. CasPlus-V3 and V4 include a combination of Cas9 variants (as further described herein) and T4-WT or T4-D219A in addition to other components described. The T4 DNA polymerase described herein is targeted by MS2. CasPlus-V3 and V4 may include subcategories based on the Cas9 variants used. Cas9 variants F916P, F916del, R919P and Q920P are referred to as V3.1, V3.2, V3.3 and V3.4 in CasPlus-V3, respectively. For CasPlus-V4, the Cas9 variants are described as V4.1, V4.2, V4.3 and V4.4, respectively. "F916del" means the deletion of the F residue at position 916. The Cas9 variants can also be used in the compositions, methods and systems of the present disclosure together with RB69 DNA polymerase, wherein the RB69 polymerase optionally comprises a mutation of D222, and wherein the mutation is optionally D222A.

[0030] As shown in the examples and accompanying drawings, the system is used to accurately model and correct mutations by generating predictable insertions / deletions formed after Cas9 cutting. The system produces insertions / deletions in a DNA repair template-free manner. The system has improved characteristics relative to other gene editing systems because CasPlus editing reduces unnecessary changes to the target site and off-target site, such as large deletions, translocations, and other chromosome rearrangements compared to standard Cas9 editing. In an embodiment, the system and method reduce the end connection mediated by microhomology. In an embodiment, insertion / deletion is instead produced by non-homologous end joining (NHEJ), which is at least partially promoted by the T4 DNA polymerase as a system component.

[0031] By designing the CasPlus system and the variant with enhanced probability of producing preferred insertion / deletion, the present disclosure includes producing patient isogenic cells with higher efficiency compared with traditional homology-directed repair (HDR) methods. The results currently provided prove the practicality of the CasPlus system and its variants and the designed gRNA for characteristics other than cutting efficiency and gene specificity, and the ability to model and correct a variety of diseases based on insertion / deletion using predictable insertion / deletion formation. Therefore, the present disclosure provides compositions and methods for producing accurate insertion and / or deletion in the guide RNA targeting fragment of chromosome. Therefore, in certain embodiments, the present disclosure is used to produce insertion / deletion. Insertion / deletion includes the insertion or deletion of 1,2,3,4 or 5 nucleotides, accompanied by changes in the complementary chain, thereby causing the insertion or deletion of 1-10 base pairs (bp), including end values. By combining with the protein complex further described herein using one or more suitable guide RNAs, insertion / deletion can include any desired changes.

[0032] In non-limiting embodiments, the insertion / deletion is produced in the protein coding fragment of the chromosome, at the splice junction, in the promoter, in the enhancer element, or in any other position where the insertion / deletion is desired, as long as the appropriate original spacer adjacent motif (PAM, proto adjacent motif) is adjacent to the position of the insertion / deletion. In embodiments, the insertion / deletion correction is a mutation associated with a disorder or disease. In embodiments, the insertion / deletion correction is a frameshift mutation, a missense mutation, or a nonsense mutation. In embodiments, the insertion / deletion changes the codon of at least one amino acid in the protein coding sequence, so the mutation in the exon can be corrected to a normal (e.g., non-disease-related) exon. In embodiments, homozygous insertion / deletion can be produced. In embodiments, the insertion / deletion correction is a harmful mutation as a component of a monogenic disease (e.g., a disease caused by a variation in a single gene). In embodiments, the monogenic disease is an X-linked disease. In non-limiting embodiments, the monogenic disease is sickle cell anemia, cystic fibrosis, Huntington disease, Tay-Sachs disease, phenylketonuria, mucopolysaccharidose, lysosomal acid lipase deficiency, glycogen storage disease, galactosemia, hemophilia A, Rett syndrome, or any form of muscular dystrophy, such as Duchenne muscular dystrophy (DMD). In non-limiting embodiments, the insertion / deletion corrects a mutation in a human dystrophin gene. In embodiments, the insertion / deletion corrects a mutation (including but not necessarily limited to a deletion) in a human dystrophin gene, which consists of one or more human dystrophin gene exons 2-10 or 45-55 (including each exon). In embodiments, the insertion / deletion corrects one or more out-of-frame mutations within an exon by generating a single base pair insertion. Therefore, the present disclosure includes exon remodeling, such as reconstructing an out-of-frame reading frame. In an embodiment, the insertion / deletion restores functional dystrophin expression in cells in which the mutation is corrected. In a non-limiting embodiment, the disclosure provides for the introduction of a 1 bp insertion in exon 43, 45, 49, 51 or 53 of the human dystrophin gene. The amino acid sequence of human dystrophin and the sequence of the gene encoding human dystrophin are known in the art, such as NCBI Gene ID: 1756, including all accession numbers therein, and NCBI Accession No. NG_012232, which are incorporated herein because they exist in the NCBI database at the effective filing date of the present application or patent.

[0033] In non-limiting embodiments, the present disclosure provides correction of gene mutations associated with cystic fibrosis. In embodiments, the present disclosure provides correction of F508del in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR). The amino acid sequence of CFTR is known in the art and can be obtained as NCBI reference sequence: NP_000483.3, the amino acid sequence of which is incorporated herein because it is present in the NCBI database at the time of the effective filing date of the present application or patent. The present disclosure includes all polynucleotide sequences encoding CFTR proteins.

[0034] In embodiments, the disclosure provides fusion proteins that promote DNA polymerases to associate with wild-type Cas nuclease variants, as further described herein. In embodiments, the fusion protein comprises an MS2 domain and a T4 DNA polymerase domain, representative sequences of variants thereof are described herein.

[0035] In embodiments, the present disclosure provides insertion / deletion generation that is more frequent relative to a control. In embodiments, a control includes insertion / deletion generation values ​​obtained by using a DNA polymerase that is not a T4 DNA polymerase or RB69 DNA polymerase comprising the mutation or the system comprising a wild-type Cas9 sequence or a protein that does not exhibit nuclease activity (e.g., a detectable protein, non-limiting examples of which are provided herein and include green fluorescent protein (GFP), but other proteins, such as mCherry, may be used).

[0036] In an embodiment, if the DNA polymerase is provided as a fusion protein, the fusion protein may comprise one or more ribosomal skipping sequences, also referred to in the art as "self-cleaving" amino acid sequences. These are typically about 18-22 amino acids long. Any suitable sequence may be used, non-limiting examples of which include: T2A, comprising the amino acid sequence: EGRGSLLTCGDVEENPGP (SEQ ID NO: 42); P2A, comprising the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 43); E2A, comprising the amino acid sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 44); and F2A, comprising the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 45).

[0037] In an embodiment, the fusion protein may include connecting amino acids (e.g., linkers) that separate one or more protein domains. The linker is typically at least two amino acids long and may include a GS sequence, but other sequences may be used. In an embodiment, the length of the linker is 3-100 amino acids. In an embodiment, the linker sequence includes or consists of a "GS" sequence. In an embodiment, the linker includes or consists of the sequence SAGGGGSGGGGSGGGGSG (SEQ ID NO: 46).

[0038] In an embodiment, the fusion proteins of the present disclosure include one or more nuclear localization signals, representative non-limiting examples of which are provided herein. Generally speaking, for eukaryotic purposes, the nuclear localization signal comprises a short sequence of one or more positively charged lysine or arginine.

[0039] In a non-limiting embodiment, the present disclosure provides a fusion protein comprising an MS2 fragment and a DNA polymerase fragment, which may further comprise the aforementioned connecting amino acids, a nuclear localization signal, and a ribosome skipping / self-cleavage sequence. A fragment refers to a portion of the protein comprising a continuous amino acid sequence. In an embodiment, the length of the fragment is sufficient to retain the function of the protein to participate in the method, and is therefore a functional fragment. In an embodiment, the fragment comprises a continuous fragment of the protein comprising a continuous 80%-99% of the amino acid sequence.

[0040] In embodiments, whether present in the fusion protein or not, the DNA polymerase is T4 DNA polymerase, but other DNA polymerases capable of filling in overhangs may be used, for example, T7 DNA polymerase may be used. We have demonstrated that the following DNA polymerases do not function in the system: DNA polymerase λ, DNA polymerase μ, DNA polymerase β, DNA polymerase 4 from yeast, DNA polymerase I from bacteria, and Klenow fragment all failed to show sufficient or any detectable function (see, e.g., Figure 1D-1E ).

[0041] In embodiments, the T4 DNA polymerase comprises the following sequence:

[0042] (SEQ ID NO:47).

[0043] Any suitable MS2 sequence can be used, which provides a binding site for the MS2 bacteriophage coat protein. [Seminars in Virology 8, 176-185 (1997), article number VI970120, the disclosure of which is incorporated herein by reference]. In an embodiment, the fusion protein of the present disclosure comprises an MS2 sequence comprising the following sequence:

[0044] MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 48).

[0045] Any suitable MS2 bacteriophage coat protein sequence can be used, including any MS2 bacteriophage coat protein sequence having 80-99.99% sequence identity to the above sequences and providing the necessary binding sites to the MS2 RNA aptamer. In an embodiment, the fusion protein comprises a first linker sequence comprising the sequence SAGGGGSGGGGSGGGGSG (SEQ ID NO: 46). In an embodiment, the fusion protein comprises a second linker sequence comprising the sequence GS.

[0046] In an embodiment, the fusion protein comprises one or more nuclear localization signals. In an embodiment, the one or more nuclear localization signals (NLS) comprise the sequence: GPKKKRKVAAA (SEQ ID NO: 49).

[0047] In an embodiment, the system of the present disclosure comprises a fusion protein comprising a continuous polypeptide containing the following in the N->C-terminal direction: an MS2 protein fragment, a first linker, a first NLS, a T4 DNA polymerase fragment, a second linker sequence, and a second NLS. This construct can also be used as a control to demonstrate the improved performance of the CasPlus variant. Representative constructs are as follows, and are further described below:

[0048]

[0049]

[0050] The MS2 sequence is shown in bold, the adapter sequence is shown in italics, the NLS sequence is shown in enlarged font, and the T4 DNA sequence is shown in bold and italics.

[0051] In an embodiment, the present disclosure provides a fusion protein, the fusion protein being encoded by a sequence comprising or consisting of the following nucleic acid sequence and / or encoding any one of the following annotated amino acid sequences:

[0052] T4-D219A protein sequence

[0053] MS2-Linker-NLS-T4-D219A-NLS

[0054]

[0055] T4-D219A DNA sequence

[0056] MS2-Linker-NLS-T4-D219A-NLS

[0057]

[0058]

[0059] RB69 DNA polymerase protein sequence

[0060] MS2-Linker-NLS-T4-D219A-NLS

[0061]

[0062] RB69 DNA polymerase DNA sequence

[0063] MS2-Linker-NLS-RB69-NLS

[0064]

[0065]

[0066] RB69-D222A protein sequence

[0067] MS2-Linker-NLS-RB69-D222A-NLS

[0068]

[0069] RB69-D222A DNA sequence

[0070] MS2-Linker-NLS-RB69-D222A-NLS

[0071]

[0072]

[0073] T7 DNA polymerase protein sequence

[0074] MS2-Linker-NLS-T7-DNA-Pol-NLS

[0075]

[0076] T7 DNA polymerase DNA sequence

[0077] MS2-Linker-NLS-T7-DNA-Pol-NLS

[0078]

[0079]

[0080]

[0081] The present disclosure includes any suitable amino acid sequence having 80-99.99% sequence identity to the above sequences and all other sequences described herein, wherein the sequence has the required DNA polymerase activity to promote NHEJ or other DNA editing and provides the required binding site for MS2 phage coat protein.

[0082] The present disclosure includes any suitable nucleic acid sequence that can be used in the present invention that encodes any of the aforementioned amino acid sequences having 80-99.99% sequence identity, wherein the amino acid sequence has the DNA polymerase activity required to promote the DNA editing and provides the required binding site for the MS2 phage coat protein.

[0083] The purpose of the fusion protein is to "tag" T4 DNA polymerase with an MS2 protein fragment. The MS2 tag is used to recruit the MS2 protein and another protein (such as a Cas enzyme) to which MS2 is attached to an RNA sequence comprising a tetraloop (such as a guide RNA) and a stem loop 2. These features protrude from the outside of the Cas9-gRNA ribonucleoprotein complex, and the distal 4 base pairs (bp) of each stem do not interact with the Cas9 amino acid side chains. The tetraloop and stem-loop 2 allow the addition of protein-interacting RNA aptamers to facilitate recruitment of the effector domain to the Cas9 complex (e.g. [Nature volume 517, pages 583–588 (2015)], the disclosure of which is incorporated herein by reference. Thus, the system is used to recruit the T4 DNA or the RB69 polymerase to a guide RNA and Cas enzyme comprising an MS2 binding domain. Other protein recruitment systems can be used, such as SunTag, a system for recruiting multiple copies of a protein to a polypeptide scaffold. [Cell. 2014 Oct 23; 159(3): 635–646, the disclosure of which is incorporated herein by reference].

[0084] In embodiments, DNA polymerase catalyzes DNA synthesis in the 5'->3' direction to produce insertions / deletions after cleavage by a Cas enzyme. In embodiments, the system inhibits microhomology-mediated end joining. In embodiments, the present disclosure provides for the generation of 1-2 base pair staggered ends with 5' overhangs, which allow for precise and predictable insertion of 1-2 nucleotides identical to the sequence 4-5 base pairs upstream of the PAM by filling in the staggered ends mediated by DNA polymerase.

[0085] In a specific and non-limiting embodiment, Cas includes Cas9, such as Streptococcus pyogenes (SpCas9). Derivatives of Cas9 are known in the art and can also be used with the DNA polymerase. Such derivatives can be, for example, enzymes that are smaller than Cas9, and / or have different protospacer adjacent motif (PAM) requirements. In a non-limiting embodiment, the Cas enzyme can be Cas12a (also known as Cpf1), or SpCas9-HF1, or HypaCas9, or xCas9, or Cas9-NG, or SpG, or SpRY.

[0086] In non-limiting embodiments, the DNA endonuclease can be a transposon-related TnpB. The reference sequence of Streptococcus pyogenes can be obtained with GenBank accession number NC_002737, and the cas9 gene is located at position 854757-858863. The Streptococcus pyogenes Cas9 amino acid sequence can be obtained with accession number NP_269215. These sequences are incorporated herein by reference, as they are provided on the priority date of this application or patent.

[0087] The Cas enzyme is provided together with one or more suitable guide RNAs, which may be referred to as "targeting RNAs" or "targeting RNAs". Representative guide RNAs and guide RNAs used in the examples are provided in Table 1. Table 1 also provides target sites corresponding to the guide RNAs.

[0088] Typically, the targeting RNA is provided to include a suitable MS2 binding site. In an embodiment, a suitable guide RNA comprises the following sequence:

[0089] Wherein bold capital letters represent the selected spacer sequence, and bold lowercase letters represent the MS2 loop to which the T4-MS2 fusion protein binds. However, the present disclosure unexpectedly reveals that the MS2 binding site is not necessary for the CasPlus system to function. Therefore, a guide RNA with or without an MS2 binding site can be provided. In an embodiment, a DNA polymerase without any MS2 binding site can be provided. Therefore, in a non-limiting embodiment, the DNA polymerase can be provided as a DNA polymerase of a non-fusion protein fragment.

[0090] Any of the components can be introduced into the cell using any suitable route and form. In an embodiment, the present disclosure provides the use of one or more plasmids or other suitable expression vectors encoding the targeting RNA and / or the protein. In an embodiment, the present disclosure provides an RNA-protein complex, such as RNAP.

[0091] In embodiments, viral expression vectors can be used to introduce one or more components of the system. Viral expression vectors can be used as naked polynucleotides, or can include viral particles. In embodiments, expression vectors include modified viral polynucleotides, such as from adenovirus, herpes virus or retrovirus, such as lentiviral vectors. In embodiments, one or more components of the CasPlus system variant can be delivered to cells using, for example, recombinant adeno-associated virus (AAV) vectors. Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome length of about 4.7 kb, including 145 nucleotides of reverse terminal repeats (ITR). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is given in Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). The cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained in ITR. Since the signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the approximately 4.3 kb within the genome (encoding replication and structural capsid proteins, rep-cap) can be replaced by foreign DNA (e.g., an expression cassette), in which the rep and cap proteins are provided in trans. The sequences located between the ITRs of the AAV vector genome are referred to herein as "payloads." Thus, recombinant AAV (rAAV) may contain up to about 4.7 kb, 4.6 kb, 4.5 kb, or 4.4 kb of unique payload sequences. After infection of target cells, protein expression and replication of the vector require the synthesis of complementary DNA strands to form a double-stranded genome. This second strand synthesis represents the rate-limiting step in transgene expression. AAV vectors are commercially available, for example from TAKARA and other commercial suppliers, and may be adapted for use in the system given the benefit of the present disclosure. In embodiments, to produce AAV vectors, plasmid vectors may encode all or some of the well-known rep, cap, and adeno-auxiliary components. In certain embodiments, the expression vector is a self-complementary adeno-associated virus (scAAV). In scAAV vectors, the payload contains two copies of the same transgenic payload in opposite orientations to each other, i.e., the first payload sequence is followed by the reverse complement of the sequence. These scAAV genomes are able to adopt a hairpin structure in which complementary payload sequences intramolecularly hybridize to each other, or to adopt a double-stranded complex in which two genome molecules hybridize to each other. Transgenic expression from such scAAV is much more efficient than transgenic expression from traditional AAV, but because the genome needs to carry two complementary copies of the payload sequence, the effective payload capacity of the vector genome is halved. Suitable scAAV vectors are commercially available, for example from CELLBIOLABS, And given the benefit of this disclosure, it may be applicable to the presently provided embodiments.

[0092] In this specification, the term "rAAV vector" is generally used to refer to a vector having only one copy of any given payload sequence (i.e., an rAAV vector is not a scAAV vector), and the term "AAV vector" is used to cover rAAV and scAAV vectors. The AAV sequences (e.g., ITRs) in the AAV vector genome can be from any AAV serotype from which a recombinant virus can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, and AAV PHP.B. The nucleotide sequences of AAV serotype genomes are known in the art. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC001401 and Srivastava et al., J. Virol., 45:555-564 {1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the complete genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least a portion of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); the AAV-11 genome is provided in Virology, 330(2):375-383 (2004); AAV PHP.B is described in Deverman et al., Nature Biotech. 34(2), 204-209, and its sequence is deposited under GenBank Accession No. KU056473.1.

[0093] In an embodiment, a non-viral delivery system can be used to introduce one or more components of the system. Non-viral tools include hydrodynamic injection, electroporation, and microinjection. Hydrodynamic injection can deliver CasPlus variants systemically to target tissues, including but not necessarily limited to the liver. In order to penetrate endothelial cells and parenchymal cells, hydrodynamic injection requires high injection volume, speed, and pressure, which limits central nervous system treatment. Electroporation and microinjection can be used for germline editing or embryo manipulation. Chemical carriers, such as lipids and nanoparticles, are widely used for delivery. Cationic lipids interact with negatively charged DNA and cell membranes to protect DNA and cell endocytosis. DNA nanoparticles, for example, are potential delivery strategies. DNA (CRISPR-gold) conjugated to gold nanoparticles composited with cationic endosome destructive polymers can deliver the CasPlus variants to animal cells.

[0094] In an embodiment, expression vectors, proteins, RNPs, polynucleotides and combinations thereof can be provided as pharmaceutical preparations. Pharmaceutical preparations can be prepared by mixing the components with any suitable pharmaceutical additives, buffers, etc. Examples of pharmaceutically acceptable carriers, excipients and stabilizers can be found in, for example, Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, the disclosure of which is incorporated herein by reference. In addition, any of a variety of therapeutic delivery agents can be used, including but not limited to nanoparticles, lipid nanoparticles (LNPs), fusosomes, exosomes, etc. In an embodiment, biodegradable materials can be used. In an embodiment, poly (lactide-co-galactosidide) (PLGA) is a representative biodegradable material, but it is expected that any biodegradable material, including but not necessarily limited to biodegradable polymers. As a substitute for PLGA, biodegradable materials can include poly (glycolide) (PGA), poly (L-lactide) (PLA) or poly (β-amino esters). In embodiments, the biodegradable material may be a hydrogel, alginate or collagen. In embodiments, the biodegradable material may include polyester, polyamide or polyethylene glycol (PEG). In embodiments, lipid-stabilized microparticles and nanoparticles may be used.

[0095] In embodiments, combinations of proteins and combinations of one or more proteins and polynucleotides described herein can be first assembled in vitro and then administered to a cell or organism.

[0096] The cells into which the system is introduced are not particularly limited, and may include postmitotic adult tissues that are considered resistant to HDR, such as heart and bone cells. The present disclosure is not necessarily limited to such cells, and may also be used with, for example, totipotent, pluripotent, multipotent or oligopotent stem cells. In an embodiment, the cell is a neural stem cell. In an embodiment, the cell is a hematopoietic stem cell. In an embodiment, the cell is a leukocyte. In an embodiment, the leukocyte belongs to the myeloid or lymphoid lineage.

[0097] In embodiments, the cell is an embryonic stem cell or an adult stem cell. In embodiments, the cell is an epidermal stem cell or an epithelial stem cell. In embodiments, the cell is a muscle precursor cell, such as a quiescent satellite cell or a myoblast, including but not necessarily limited to a skeletal myoblast and a cardiac myoblast.

[0098] In some instances, lymphocytes are T cells. In some instances, modified T cells are also modified so that they express chimeric antigen receptors (CAR). In embodiments, cells are natural killer (NK) or natural killer T cells, which can also be modified to express CAR.

[0099] As is known in the art, T cells can be modified by using a classical Cas system to increase safety by knocking out PDCD1, TRBC1, TRBC2 and TRAC. In some embodiments, the system is used to produce insertions / deletions in one or more of the genes PDCD1, TRBC1, TRBC2 and TRAC in T cells. The disclosure shows that translocation events can be suppressed using the system. The Cas system previously used to modify these genes increases the risk of translocation. The disclosure shows that the use of the system reduces the risk of translocation, thus providing a method for producing modified cells more safely, including but not necessarily modified T cells to be used in the form of CAR. In embodiments, the use of the CasPlus system reduces balanced or unbalanced translocations. In embodiments, the use of the CasPlus system reduces intrachromosomal or interchromosomal translocations. In embodiments, the use of the CasPlus system reduces large deletions caused by previous systems. In embodiments, large deletions are deletions of at least 500 nucleotides.

[0100] Therefore, the present invention provides the use of the CasPlus system to produce insertions / deletions, as an alternative to previously available Cas systems or other targeted nucleases in which knockout or other destruction or modification of genes is desired but may produce the risk of translocation. Therefore, in embodiments, the present disclosure provides the use of the CasPlus system as an alternative to any other guide-guided or other targeted nucleases for simultaneously modifying one or more loci. In embodiments, the present disclosure provides an alternative to modification using any type of Cas enzyme, zinc finger nuclease or transcription activator-like effector nuclease (TALEN) or a transposon-based DNA editing system. In embodiments, the CasPlus system is used to modify at least two genetic positions while reducing the risk of translocation. Therefore, the CasPlus system can be used with 2, 3, 4 or more guide RNAs simultaneously or sequentially to modify more than one locus while reducing the risk of translocation events.

[0101] In embodiments, the disclosure includes obtaining cells from an individual, modifying the cells in vitro using a system as described herein, and reintroducing the cells or their progeny into an individual or an immune-matched individual to prevent and / or treat a condition, disease or disorder as described above. In embodiments, the modified cells as described herein are autologous cells. In embodiments, the cells are mammalian cells. Therefore, the disclosure is applicable to a wide range of human, veterinary, experimental animal and cell culture uses.

[0102] The following examples are intended to illustrate but not limit the present disclosure.

[0103] Example

[0104] Identification of T4 and RB69 DNA polymerases as proteins that facilitate CasPlus editing.

[0105] The T4 DNA polymerase-mediated CasPlus editing system can enhance the filling of 5' overhangs generated by Cas9, leading to an increase in 1-bp insertions, while inhibiting the annealing of microhomology (MH) at double-strand break (DSB) sites, thereby reducing deletions generated by the microhomology-mediated end joining (MMEJ) repair pathway ( Figure 1A We investigated whether overexpression of other phage-derived DNA polymerases would affect the results of Cas9-mediated insertion / deletion in the tdTomato reporter cell line. We first constructed an expression vector for MS2-tagged DNA polymerase optimized for human codons. Subsequently, we transfected the tdTomato reporter cell line with vectors expressing Cas9, GFP, or tdTomato-sgRNA alone or in combination with different MS2-tagged DNA polymerases. Transfected cells were sorted into cells expressing only GFP (tdTomato - / GFP + ) or expressing both tdTomato and GFP (tdTomato + / GFP + ) for genomic DNA isolation and sequencing ( Figure 1B ).tdTomato - / GFP + High-throughput sequencing (HTS) of the population showed that T4 and RB69 DNA polymerases (which have 74% amino acid similarity (27) ) resulted in an approximately 6-fold increase in the frequency of 2-bp insertions at the expense of the frequency of deletions ( Figure 1C ). This effect was not observed with overexpression of T7 DNA polymerase (28) tdTomato + / GFP + HTS of the population showed that the insertion / deletion profiles were similar in all treatment groups. Further analysis of the insertion patterns indicated that tdTomato - / GFP + >95% of 2-bp insertions in the population were template-dependent ( Figure 1D We confirmed the expression of all DNA polymerases expressed in the tdTomato reporter cell line by Western blot analysis ( Figure 1E ). Taken together, these results indicate that RB69 and T4 DNA polymerases are beneficial for CasPlus editing.

[0106] T4 DNA polymerase mutant D219A (T4-D219A) improves the efficiency of T4 DNA polymerase-mediated CasPlus editing.

[0107] Given that the efficiency of insertions generated by CasPlus editing is highly dependent on the efficiency of filling in 5' overhangs by T4 DNA polymerase, we analyzed whether enhancement of the 5'→3'-polymerase activity or reduction of the 3'→5'-exonuclease activity of T4 DNA polymerase could further improve CasPlus editing efficiency ( Figure 2A T4 DNA polymerase is multifunctional, capable of replicating DNA and proofreading misincorporated nucleotides using its exonuclease domain ( Figure 2B ). The 3'-5' exonuclease activity of T4 DNA polymerase is one of the important determinants of its activity (29)Many mutant strains of T4 bacteriophage contain T4 DNA polymerases with defective or hyperactive exonuclease domains. In the present disclosure, we constructed two T4 mutants associated with reduced DNA mutation rate (W213Y and W844S), five mutants associated with increased DNA mutation frequency (G82D, D112A, D219A, E191A-D324G, and G694S), and an N-terminal truncation mutant lacking the 3'-5' exonuclease domain (deletion 1-377aa). (24-26) ( Figure 2B To assess the efficiency of the promoted insertion, we tested target site (TS) 11, which resulted in a relatively small increase in 1-bp insertions upon overexpression of wild-type T4 DNA polymerase (T4-WT). Strikingly, coexpression of mutant T4-D219A increased 1-bp insertions at TS11 by 2.4-fold compared to WT-T4 ( Figure 2C In contrast, overexpression of other T4 mutants resulted in a reduction of the 1-bp insertion at TS11 compared with T4-WT.

[0108] We further tested the activity of the T4-D219A mutant at additional genomic loci. Compared to T4-WT, the T4-D219A mutant caused an additional 1.8- to 2.8-fold increase in 1-bp insertions in all three additional tested genomic loci ( Figure 2D ). The T4-D219A mutant also resulted in a 2-fold increase in 1-bp and 2-bp insertions at TS17, and a 1.8- and 1.7-fold increase in 3-bp and 1-bp insertions at TS18 compared to T4-WT ( Figure 2E At TS26, although T4-WT and Cas9 failed to promote 1bp insertion, T4-D219A and Cas9 induced a 2.3-fold increase in 1bp insertion compared with Cas9 alone ( Figure 2F ).

[0109] Cas12a (also known as Cpf1) is another Cas nuclease that can generate 5' overhangs with 5-8 nucleotides. (30) We tested whether T4 DNA polymerase could fill in the overhangs induced by Cas12a, resulting in insertions of 5–8 nucleotides ( Figure 2G ). In contrast, the cleavage site of Cas12a is located at the distal end of the PAM sequence (18-23 bp from PAM), so Cas12a can re-cleave the target site to produce indels or indels with 5-8 nucleotide repeats. (31). Therefore, we calculated the frequency of editing products that contained insertions but not duplications. HTS results showed that in the absence of T4 DNA polymerase, Cas12a produced editing products with <2% insertions. In contrast, in the presence of T4-WT or T4-D219A, Cas12a produced insertion frequencies of 17% or 39%, respectively ( Figure 2H ). These results indicate that T4-D219A exhibits improved CasPlus editing efficiency compared to T4-WT.

[0110] RB69 DNA polymerase mutant D222A (RB69-D222A) improves the efficiency of RB69 DNA polymerase-mediated CasPlus editing.

[0111] Previous sequence analysis showed that T4 DNA polymerase residue Asp-219 is similar to Asp-222 in wild-type RB69 (RB69-WT) DNA polymerase from RB69 bacteriophage (32) Therefore, we investigated the activity of the RB69-D222A mutant at local genomic loci. Compared with RB69-WT, RB69-D222A increased the 2-bp insertion at the tdTomato locus ( Figure 3A RB69-D222A also resulted in a 2.3-fold, 3.9-fold, and 2.2-fold increase in 1-bp insertions at TS2, TS11, and TS12, respectively, compared to RB69-WT ( Figure 3B ). Therefore, both T4-D219A and RB69-D222A mutations can further improve the 1-bp insertion editing efficiency of CasPlus in human cells.

[0112] At Cas9 target sites where Cas9-WT and T4-WT predominantly produced deletions, the combination of Cas9 variants with T4 DNA polymerase enhanced 1-bp insertions.

[0113] Given that CasPlus editing is associated with DSB ends with 5' overhangs, its editing efficiency is limited by the number and type of staggered ends generated by Cas9 editing. Most DSBs induced by Cas9-WT are blunt ends, whereas some Cas9 variants can be rationally engineered to favor the generation of 1-bp overhangs. (33) We analyzed whether combining these rationally engineered Cas9 variants with T4 DNA polymerase could further enhance the frequency of 1-bp insertions ( Figure 4A-4B). To test this, we used TS11 as a target and transfected cells with rationally engineered Cas9 variants alone or in combination with T4-WT. The present disclosure shows that although the editing efficiency of Cas9 variants at TS11 was reduced compared to wild-type Cas9 (Cas9-WT), the individual Cas9 variants F916P, F916del, R919P or Q920P produced approximately 16% of products with 1-bp insertions, while Cas9-WT alone produced 4% of 1-bp insertions ( Figure 4C ). Interestingly, the combination of Cas9 variants F916P, F916del, R919P, or Q920P with T4-WT produced approximately 44% to 55% 1-bp insertions, while the combination of Cas9-WT with T4-WT produced approximately 15% edits with 1-bp insertions ( Figure 4D ). These results suggest that the combination of Cas9 variants with T4 DNA polymerase is able to enhance 1-bp insertion. Given that both the deletion of Phe-719 and the mutation of Phe-719 to Pro-719 increased 1-bp insertion in CasPlus editing, we chose to focus the examples described subsequently on the Phe-719 mutation.

[0114] Our subsequent experiments focused on five target sites that initially showed a modest increase in 1-bp insertions in the presence of Cas9-WT and T4-WT. We found that in the presence of T4-D219A, the Cas9 variants F916P and F916del resulted in an average increase of 4.3-fold or 5.1-fold, respectively, in 1-bp insertions at the five target sites compared to the Cas9 variants alone. Figures 4E-4F ). These results suggest that T4 DNA polymerase can enhance 1-bp insertions when combined with Cas9 variants at target sites where Cas9-WT and T4-WT predominantly produce deletions. Overall, the new strategy of combining Cas9 variants with T4 DNA polymerase expands their target site range for 1-bp insertion editing outcomes.

[0115] Combination of Cas9 variants and T4 DNA polymerase enhances the generation of longer inserts (2 to 4 bp)

[0116] Our previous experiments showed that the combination of engineered Cas9 variants with T4 DNA polymerase can increase the frequency of 1-bp insertions at Cas9 target sites where Cas9-WT and T4-WT primarily generate deletions. Therefore, we analyzed whether the same combination of Cas9 variants with T4 DNA polymerase can increase the frequency of longer insertions (e.g., 2 to 4-bp insertions) at Cas9 target sites where Cas9-WT and T4-WT initially generate primarily 1-bp insertions. Figure 5AWe focused on the previously described tdTomato loci where Cas9-WT and T4-WT predominantly generate 2-bp insertions to determine whether the combination of Cas9 variants with T4 DNA polymerase could increase the frequency of 3-bp or longer insertions. HTS showed that in the presence of T4 DNA polymerase, Cas9 variants F916P, F916del, and Q920P resulted in a significant increase in 3-bp insertions compared to Cas9-WT, whereas the Cas9 variants alone did not alter the frequency of 3-bp insertions ( Figure 5B-5C ).

[0117] Next, we investigated the ability of Cas9-F916P and Cas9-F916del to generate longer insertions at other genomic sites. We used TS5, TS17, and TS18, which primarily generated 1-bp, 2-bp, and 3-bp insertions, respectively, with Cas9-WT and T4-WT. At TS5, Cas9-F916P and Cas9-F916del promoted the generation of 2-bp or 3-bp insertions when combined with T4 DNA polymerase; at TS17 and TS18, the Cas9 variants promoted the generation of 3-bp and 4-bp insertions when combined with T4 DNA polymerase ( Figure 5D ). These findings led us to conclude that the combination of Cas9 variants with T4 DNA polymerase can enhance the generation of longer inserts (2 to 4 bp).

[0118] To illustrate the versatility of the T4 DNA polymerase-mediated CasPlus system, we divided it into four versions. CasPlus-V1 is a combination of Cas9-WT and T4-WT. CasPlus-V2 labeled a combination of Cas9-WT and T4-D219A. CasPlus-V3 and V4 use a combination of Cas9 variants and T4-WT or T4-D219A, respectively. CasPlus-V3 and V4 are further divided into subcategories based on the Cas9 variants used. Cas9 variants F916P, F916del, R920P, and Q920P are named V3.1, V3.2, V3.3, and V3.4, respectively, in CasPlus-V3; or V4.1, V4.2, V4.3, and V4.4, respectively, in CasPlus-V4 ( Figure 5E ). As mentioned above, all T4 DNA polymerases carry the MS2 tag.

[0119] The CasPlus system effectively suppresses large on-target deletions.

[0120] A major problem with conventional CRISPR / Cas9 technology in clinical and preclinical trials is its potential to generate uncontrolled and unexpected large deletions and complex chromosomal rearrangements at the Cas9 target site. (5、34) These large deletions are often caused by long-range end resections due to Cas9-induced DSBs ( Fig. 6A ). Our HTS data using ~300-bp PCR amplicons indicated that CasPlus editing primarily enhances insertions at the expense of small deletions (<100 bp). We analyzed whether CasPlus editing could also suppress the generation of large deletions (>500 bp) by filling in or binding to DSB-induced ends prior to long-range end resection ( Fig. 6A ). To test this, we assessed the presence of large deletions at the X-linked DMD locus. We used male iPS cells (iPSCs) to deliver guide RNAs targeting TS10 or TS9 on DMD exon 51 or 53, respectively. These guide RNAs were tested in combination with Cas9 and with the CasPlus system. Previous reports have shown that repair of Cas9-induced DSBs results in an asymmetric distribution of on-target indels, favoring changes in PAM-distal or 5' regions. (35) Therefore, we designed two primer sets to amplify 1-2.0 kb PAM distal or proximal regions of the target site from the edited cell pool ( Figure 6B and 6D ). From the PAM-distal region, Cas9-edited cells were amplified, electrophoresed on gels, and imaged. We observed several lower bands that appeared only in Cas9-edited cells in our PCR gels, representing deletions of approximately 450 bp and 1.3 kb at TS10 and TS9, respectively. ( Figure 6C and 6E Next, we amplified the ~5-kb region around the DMD exon 51 and 53 target sites from the edited iPSC library and sequenced the PCR amplicons using PacBio sequencing technology. Up to 23.0% of PacBio reads contained deletions of 0.2-3 kb around the cut site of exon 51 in Cas9-edited cells ( Fig. 6Fand Table 2). We did not observe this effect in untreated cells (approximately 2.0%) or in cells edited with CasPlus-V1 (approximately 3.2%) or CasPlus-V2 (approximately 3.5%). In untreated cells, we detected a deletion of approximately 3 kb around DMD exon 53 in 13.2% of PacBio reads. This result may be due to technical issues introduced during PCR amplification, as similar-sized 3-kb deletions were observed in all samples tested (Cas9 (11.1%); CasPlus-V1 (9.4%); CasPlus-V2 (14.8%)). At DMD exon 53, Cas9 greatly increased the number of reads with 0.2-3.5 kb deletions around the cleavage site compared with untreated cells or cells edited with CasPlus-V1 or CasPlus-V2 (Cas9 (48.9%); CasPlus-V1 (9.5%); CasPlus-V2 (17.4%)) ( Figure 6G and Table 2). Thus, CasPlus-V1- and CasPlus-V2-mediated editing efficiently suppressed on-target large deletions.

[0121] Enhanced correction of DMD exon 52 deletion in iPSCs by CasPlus editing.

[0122] CasPlus system editing can enhance 1-bp insertions at the expense of small or large deletions at the Cas9 target site, making it a valuable tool for gene knockout and treatment of diseases caused by 3n-1 indels. Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations in the dystrophin gene that lead to fatal degeneration of heart and skeletal muscle. (36) Previously, we corrected DMD mutations by CRISPR / Cas9-mediated single-site editing at RNA splice sites or by excision of exons by double cleavage. (21,37) . Both strategies aim to excise exons to correct the open reading frame. However, single-site editing is limited to RNA splice sites, and double cutting may increase the risk of unwanted large deletions, translocations, and other chromosomal rearrangements. With this in mind, we tested the effectiveness of CasPlus-mediated single-site editing to correct DMD mutations. We initially generated an iPSC model with DMD exon 52 deletion using CRISPR / Cas9 gene editing. We analyzed whether precise reinsertion of 1-bp at the 3' end of exon 51 or the 5' end of exon 53 could effectively repair the dystrophin gene in iPSCs with exon 52 deletion ( Fig. 7A We designed comprehensive guide RNA libraries containing NGG PAM for both target regions ( Figure 7B) and tested their editing efficiency in HEK293T cells. We found that TS10 had a slightly higher editing efficiency than TS27. We also found that TS9 and TS28 exhibited much higher editing efficiency than other guide RNAs targeting exon 53. Therefore, we selected TS10 and TS9 to correct DMD exon 52 deletions in iPSCs. HTS showed that CasPlus-V2 had the highest frequency of both 1-bp insertions and corrected reading frames compared to CasPlus-V1 or Cas9 alone ( Figure 7C ). We further differentiated the edited iPSC pool from iPSC single clones (SC) using 1-bp insertions into cardiomyocytes (iCMs). For each target site, we designed a set of RT-PCR primers to reveal the profile of small insertions / deletions and another set of primers to detect exon skipping caused by larger deletions. HTS results showed that the proportion of mRNA alleles with 1-bp insertions and corrected reading frames was the highest in CasPlus-V2-edited iCMs ( Fig.7D We confirmed that when TS9 and TS10 were used to target DMD exons 51 and 53, large deletions occurred in cells edited with Cas9 alone ( Figure 6B-6E We analyzed whether genes with large deletions had lost part or all of the target exon, thereby inducing target exon skipping at the mRNA level. Sanger sequencing results confirmed that entire exon 51 and 53 skipping occurred in iCMs edited with Cas9 alone ( Fig. 7E ). Next, Western blot analysis showed that dystrophin expression was restored in the edited iCM pool. CasPlus-V1 and CasPlus-V2 treatments had higher dystrophin expression compared to the control treatment using Cas9 alone. ( Figure 7F ).

[0123] Exogenous template-independent correction of the CFTR F508del mutation by sequential CasPlus editing.

[0124] CasPlus editing-induced insertions that are independent of exogenous templates can be used to precisely correct genetic diseases caused by 1- to 3-bp deletions. Cystic fibrosis is an autosomal recessive disease that involves functional defects in mucus- and sweat-producing cells and severely affects multiple organs, especially the lungs. It is caused by mutations in the gene that produces the cystic fibrosis transmembrane conductance regulator (CFTR) protein. (38,39) The most common CFTR mutation is a 3-bp deletion resulting in the loss of a phenylalanine at position 508 (F508del), which accounts for approximately 70–80% of all pathogenic mutations in CFTR. (40) ( Fig. 8ADrugs have been developed to improve clinical symptoms and prevent complications in CFTR patients. (41) However, the possibility of targeted gene therapy at the DNA level has hardly been explored. Here, we employed sequential CasPlus editing to precisely correct the CFTR-F508del mutation. We initially generated a cellular model of CFTR-F508del in HEK293T cells using HDR-mediated knock-in ( Figure 8B Based on the sequences flanking CFTR-F508del, we tested four potential outcomes for restoring gene expression by CasPlus editing: a CFTR protein with a missense amino acid (one-step editing), insertion of AT in the first step and T in the second step, insertion of T in the first step and TT in the second step, and three-step introduction of TTT, which would restore expression of WT CFTR protein ( Figure 8C We designed guide RNAs for sequential editing that initially targeted the CFTR-F508del allele (TS32) and then inserted an intervening AT (TS34) or T, or included T (TS33) and / or TT (TS35 and TS36) to produce the desired edits ( Fig.8D ). We first delivered a vector expressing the guide RNA TS32 in combination with Cas9-NG-WT, Cas9-NG-F916P, or CasPlus editors into HEK293T cells with a homozygous CFTR-F508del mutation. We observed that using the guide RNA (TS32), CasPlus-V1 and CasPlus-V2 or CasPlus-V3.1 and CasPlus-V4.1 had higher 1- and 2-bp insertion frequencies relative to Cas9-NG-WT or Cas9-NG-F916P ( Fig. 8E ). Next, we tested two-step sequential CasPlus editing. We confirmed that the combination of CasPlus-V1, CasPlus-V2, CasPlus-V3.1, and CasPlus-V4.1 with guide RNAs (TS32) and (TS34) produced 8%, 10%, 14.5%, and 14.6% of edits with 3-bp insertions, respectively. On the other hand, the combination of CasPlus-V1, CasPlus-V2, CasPlus-V3.1, and CasPlus-V4.1 with guide RNAs TS32 and TS33 produced 3.3%, 4.5%, 5%, and 6% of edits with 3-bp insertions, respectively. Figure 8F-8G). We concluded that the combination of CasPlus-V3.1 or V4.1 with guide RNAs TS32 and TS34 exhibited the highest percentage of 3-bp insertions. In addition, cells treated with CasPlus-V3.1 or CasPlus-V4.1 in combination with guide RNAs TS32 and TS34 had an editing profile in which approximately 30-40% of the indels were 1-bp insertions. Therefore, we analyzed whether the combination of guide RNAs TS32, TS33, and TS34 could further enhance the generation of 3-bp insertions. We delivered the CasPlus system with guide RNA combinations of TS32, TS33, and TS34 into homozygous CFTR-F508del cells and confirmed that CasPlus-V1, V2, V3.1, and V4.2 induced 16%, 19%, 17%, and 18% of edits with 3-bp insertions, respectively ( Figure 8H ). We also tested three-step sequential CasPlus editing using guide RNAs TS32, TS34, and TS35. The results showed that CasPlus-V2 exhibited the highest 3-bp insertion percentage (12.8%). Analysis of the 3-bp insertion pattern after sequential CasPlus editing in combination with different guide RNAs demonstrated that >90% of the 3-bp insertions were edits of corrected CFTR with silent mutations, rather than WT CFTR ( Figures 8I-8J Based on the above results, we concluded that sequential CasPlus editing can effectively and accurately correct the CFTR-F508del mutation.

[0125] CasPlus editing suppresses on-target chromosomal translocation between two chromosomes.

[0126] Chromosomal translocation occurs when two DSBs occur simultaneously on two chromosomes. Fig. 9A To investigate whether CasPlus editing could reduce chromosomal translocations, we recapitulated a previously described translocation event between the CD74 and ROS1 genes in HEK293T cells. (42) ( Fig. 9B We amplified the breakpoint junction regions on the fusion chromosomes by PCR and determined the translocation efficiency. We detected and validated ROS1-CD74 and CD74-ROS1 translocations induced by Cas9 and CasPlus editing ( Fig. 9C Compared with Cas9 editing, the translocation frequency of CasPlus-V1 was approximately 5-fold lower, and the translocation frequency of CasPlus-V2 was approximately 2-fold lower ( Fig. 9C and 9D ). Compared with Cas9 editing, CasPlus-V1 and -V2 editing resulted in higher insertion frequencies at the ROS1 and CD74 individual sites ( Fig.9EWe observed a similar trend in chromosomal translocation suppression in iPSCs ( Figure 9F-9H ). CasPlus editing suppresses on-target chromosomal translocations between multiple chromosomes.

[0127] We next investigated chromosomal translocations between the genes PDCD1, TRBC1, TRBC2, and TRAC (located on chromosomes 2, 7, and 14) in HEK293T cells induced by three gRNAs used in previous T cell-based clinical trials. (6,7) ( Fig. 10A and Fig.11A CasPlus-V1 resulted in a 2.5- to 4.5-fold reduction in all types of translocations tested between these four genes ( Fig. 10B and 10C as well as Fig. 11B and 11C ). CasPlus-V1 editing induced similar knockout efficiencies at these four individual sites compared to Cas9 editing ( Fig. 10D ). CasPlus-V2 had similar knockout effects as CasPlus-V1 but was less efficient in suppressing translocations. Therefore, our proof-of-concept results suggest that CasPlus editing significantly suppresses Cas9-mediated on-target chromosomal translocations and is a potentially safer approach for T cell-related therapies.

[0128] References - This list of references does not imply that any reference is essential to patentability.

[0129] 1.M.Jinek et al.,A programmabledual-RNA-guided DNA endonuclease inadaptive bacterial immunity.Science 337,816-821(2012).

[0130] 2. M. Jinek et al., RNA-programmed genome editing in human cells. Elife2, e00471 (2013). 3. L. Cong et al., Multiplex genome engineering using CRISPR / Cassystems. Science 339, 819-823 (2013).

[0131] 4.P.Mali et al.,RNA-guided human genome engineering via Cas9.Science339,823-826(2013).

[0132] 5.M.Kosicki,K.Tomberg,A.Bradley,Repair of double-strand breaksinduced by CRISPR-Cas9 leads to large deletions and complexrearrangements.Nat Biotechnol 36,765-771(2018).

[0133] 6.A.D.Nahmad et al.,Frequent aneuploidy in primary human T cellsafter CRISPR-Cas9cleavage.Nat Biotechnol,(2022).

[0134] 7.E.A.Stadtmauer et al.,CRISPR-engineered T cells in patients withrefractory cancer.Science367,(2020).

[0135] 8.M.L.Leibowitz et al.,Chromothripsis as an on-target consequence ofCRISPR-Cas9 genome editing.Nat Genet 53,895-905(2021).

[0136] 9.F.Uddin,C.M.Rudin,T.Sen,CRISPR Gene Therapy:Applications,Limitations,and Implications for the Future.Front Oncol 10,1387(2020).

[0137] 10.X.Shi et al.,Cas9 has no exonuclease activity resulting instaggered cleavage with overhangs and predictabledi-and tri-nucleotide CRISPRinsertions without template donor.Cell Discov 5,53(2019).

[0138] 11.H.H.Y.Chang,N.R.Pannunzio,N.Adachi,M.R.Lieber,Non-homologous DNAend joining and alternative pathways to double-strand break repair.Nat RevMol Cell Biol 18,495-506(2017).

[0139] 12.D.D.G.Owens et al.,Microhomologies are prevalent at Cas9-inducedlarger deletions.Nucleic Acids Res 47,7402-7417(2019).

[0140] 13.M.Kosicki et al.,Cas9-induced large deletions and small indels arecontrolled in a convergentfashion.Nat Commun 13,3422(2022).

[0141] 14.M.W.Shen et al.,Predictable and precise template-free CRISPRediting of pathogenicvariants.Nature 563,646-651(2018).

[0142] 15.F.Allen et al.,Predicting the mutations generated by repair ofCas9-induced double-strandbreaks.Nat Biotechnol,(2018).

[0143] 16.R.T.Leenay et al.,Large dataset enables prediction of repair afterCRISPR-Cas9 editing inprimary T cells.Nat Biotechnol 37,1034-1037(2019).

[0144] 17.A.M.Chakrabarti et al.,Target-Specific Precision of CRISPR-Mediated Genome Editing.Mol Cell 73,699-713 e696(2019).

[0145] 18.K.F.O'Brien,L.M.Kunkel,Dystrophin and muscular dystrophy:past,present,and future.Mol Genet Metab 74,75-88(2001).

[0146] 19.F.Muntoni,S.Torelli,A.Ferlini,Dystrophin and mutations:one gene,several proteins,multiple phenotypes.Lancet Neurol 2,731-740(2003).

[0147] 20.R.Adorisio et al.,Duchenne Dilated Cardiomyopathy:CardiacManagement from Preventionto Advanced Cardiovascular Therapies.J Clin Med 9,(2020).

[0148] 21.C.Long et al.,Correction of diverse muscular dystrophy mutationsin human engineered heartmuscle by single-site genome editing.Sci Adv 4,eaap9004(2018).

[0149] 22.C.Long et al.,Postnatal genome editing partially restoresdystrophin expression in a mousemodel of muscular dystrophy.Science 351,400-403(2016).

[0150] 23.C.Long et al.,Prevention of muscular dystrophy in mice by CRISPR / Cas9-mediated editingof germline DNA.Science 345,1184-1188(2014).

[0151] 24.L.J.Reha-Krantz,Amino acid changes coded by bacteriophage T4 DNApolymerase mutatormutants.Relating structure to function.J Mol Biol 202,711-724(1988).

[0152] 25.L.J.Reha-Krantz,Regulation of DNA polymerase exonucleolyticproofreading activity:studies of bacteriophage T4"antimutator"DNApolymerases.Genetics 148,1551-1557(1998).26.A.K.Abdus Sattar,T.C.Lin,C.Jones,W.H.Konigsberg,Functional consequences andexonuclease kinetic parameters ofpoint mutations in bacteriophage T4 DNA polymerase.Biochemistry 35,16621-16629(1996).

[0153] 27.H.K.Dressman,C.C.Wang,J.D.Karam,J.W.Drake,Retention of replicationfidelity by aDNA polymerase functioning in a distantly relatedenvironment.Proc Natl Acad Sci U S A 94,8042-8046(1997).

[0154] 28.K.Hori,D.F.Mark,C.C.Richardson,Deoxyribonucleic acid polymerase ofbacteriophageT7.Characterization of the exonuclease activities of the gene 5protein and the reconstitutedpolymerase.J Biol Chem 254,11598-11604(1979).

[0155] 29.T.L.Capson et al.,Kinetic characterization of the polymerase andexonuclease activities ofthe gene 43 protein of bacteriophage T4.Biochemistry31,10984-10994(1992).

[0156] 30.B.Zetsche et al.,Cpf1 is a single RNA-guided endonuclease of aclass 2 CRISPR-Cas system.Cell 163,759-771(2015).

[0157] 31.D.Kim et al.,Genome-wide analysis reveals specificities of Cpf1endonucleases in humancells.Nat Biotechnol 34,863-868(2016).

[0158] 32.M.Hogg,W.Cooper,L.Reha-Krantz,S.S.Wallace,Kinetics of errorgeneration inhomologous B-family DNA polymerases.Nucleic Acids Res 34,2528-2535(2006).

[0159] 33.J.Shou,J.Li,Y.Liu,Q.Wu,Precise and Predictable CRISPR ChromosomalRearrangementsReveal Principles of Cas9-Mediated Nucleotide Insertion.MolCell 71,498-509 e494(2018).

[0160] 34.H.Y.Shin et al.,CRISPR / Cas9 targeting events cause complexdeletions and insertions at 17sites in the mouse genome.Nat Commun 8,15464(2017).

[0161] 35.B.Farboud,A.F.Severson,B.J.Meyer,Strategies for Efficient GenomeEditing UsingCRISPR-Cas9.Genetics 211,431-457(2019).

[0162] 36.K.P.Campbell,S.D.Kahl,Association of dystrophin and an integralmembrane glycoprotein.Nature 338,259-262(1989).

[0163] 37.Y.Zhang et al.,CRISPR-Cpf1 correction of muscular dystrophymutations in humancardiomyocytes and mice.Sci Adv 3,e1602814(2017).

[0164] 38.B.P.O'Sullivan,S.D.Freedman,Cystic fibrosis.Lancet 373,1891-1904(2009).

[0165] 39.S.D.Patel,T.R.Bono,S.M.Rowe,G.M.Solomon,CFTR targeted therapies:recentadvances in cystic fibrosis and possibilities in other diseases of theairways.Eur Respir Rev 29,(2020).

[0166] 40.P.B.Davis,Cystic fibrosis since 1938.Am J Respir Crit Care Med173,475-482(2006).41.M.M.Rafeeq,H.A.S.Murad,Cystic fibrosis:currenttherapeutic targets and future approaches.J Transl Med 15,84(2017).

[0167] 42.P.S.Choi,M.Meyerson,Targeted genomic rearrangements using CRISPR / Cas technology.Nat Commun 5,3728(2014).

[0168] 43.F.A.Ran et al.,Genome engineering using the CRISPR-Cas9 system.NatProtoc 8,2281-2308(2013).

[0169] 44.L.Pinello et al.,Analyzing CRISPR genome-editing experiments withCRISPResso.Nat Biotechnol 34,695-697(2016).

[0170] 45.Statistical Genomics.Methods and Protocols.Anticancer Res 36,3224(2016).

[0171] 46. ​​H. Li, Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094-3100 (2018).

[0172] Materials and methods

[0173] Plasmids

[0174] The vector pSpCas9(BB)-2A-GFP(PX458) (Addgene plasmid #48138) contains the human codon-optimized SpCas9 gene with 2A-GFP and the sgRNA backbone and was purchased from Addgene. pLentiV-SgRNA-tdTomato-P2A-BlasR (Addgene plasmid #110854) and EF1A-CasRx-2A-EGFP (Addgene plasmid #109049) were gifts from Dr. Lukas Dow and Dr. Patrick Hsu, respectively. To construct a lentiviral vector expressing tdTomato-d151A, the tdTomato-d151A gene was synthesized by Integrated DNA Technologies (IDT). First, it was cloned into the vector p3xFlag-CMV-10, and then CMV-10-tdtomato-d151A was cloned into pLentiv-SgRNA-tdTomato-P2A-BlasR using MluI and BamHI restriction sites. For DNA polymerase cloning, the coding sequences of DNA polymerase 4, DNA polymerase I, Klenow fragment, T4 DNA polymerase, RB69 DNA polymerase, and T7 DNA polymerase were codon-optimized for human cell expression using the Genewiz codon optimization tool. For each DNA polymerase, an expression cassette containing the polymerase, MS2 (MS2 bacteriophage coat protein) and hemagglutinin (HA) tags, two copies of the nuclear localization sequence (NLS), and a flexible linker was synthesized by Genewiz and cloned into EF1A-CasRx-2A-EGFP by Gibson assembly. Mutations in T4 DNA polymerase and RB69 DNA polymerase were introduced into vectors EF1A-MS2-T4-DNA-polymerase-2A-EGFP and EF1A-MS2-RB69-DNA-polymerase-2A-EGFP, respectively, by Gibson assembly. Cas9 mutations were generated in the backbone pSpCas9(BB)-2A-GFP (PX458) by Gibson assembly. (43)Guide RNA cloning was performed using the CRIPSR plasmid instructions. All guide RNA sequences are listed in Table 1. All sequences synthesized for tdTomato-d151A or DNA polymerase cloning are listed in Table 3.

[0175] Cell lines

[0176] Generation of HEK293T cell lines containing the tdTomato-d151A reporter. To generate a stable tdTomato-d151A reporter cell line in HEK293T cells, we co-transfected the pLentiV vector expressing tdTomato-d151A and the lentiviral helper plasmids psPAX2, pMD2G, and pEGFP into HEK293T cells. Single cells expressing GFP were isolated in 96-well plates 72 hours after transfection and genotyped 2 weeks later. Positive clones were then stored and expanded for subsequent experiments.

[0177] Generation of HEK293T cells containing homozygous CFTR-F508del mutations. HEK293T cell lines containing homozygous CFTR-F508del mutations were generated by HDR-mediated gene editing. The DNA template for CFTR-F508del knock-in was synthesized by IDT. To generate mutant HEK293T cell lines, the DNA template was co-transfected with vectors expressing Cas9, GFP, and TS3. Single cells expressing GFP were isolated in 96-well plates 72 hours after transfection and genotyped 2 weeks later. Positive clones containing homozygous CFTR-F508del mutations were stored and expanded for subsequent experiments. The knock-in templates are shown in Table 3. The sequence of TS3 is shown in Table 1.

[0178] Generation of male iPS cells containing DMD exon 52 deletions. Male iPSCs were electroporated with vectors expressing Cas9, GFP, and a pair of specific guide RNAs for the deletion (DMD-Ex52-g1 and DMD-Ex52-g2, see Table 1). Single cells expressing GFP were isolated in 96-well plates 72 hours after transfection and genotyped 2 weeks later. Positive clones containing DMD exon 52 deletions were stored and expanded for subsequent experiments.

[0179] Sample preparation, DNA isolation, and PCR amplicon preparation for deep sequencing

[0180] Transfection and sorting of HEK293T cells. According to the manufacturer's instructions, HEK293T cells were transfected using Lipofectamine 2000 transfection reagent (ThermoFisher LifeTech). 72 hours after transfection, cell sorting was performed by the FlowCytometry Core Facility (FlowCytometry Core Facility) of New York University Grossman Medical Center (New York University Grossman Medical Center). In short, HEK293T cells were co-transfected with a vector expressing Cas9, sgRNA targeting different genomic sites, GFP, and a variety of DNA polymerases. 72 hours after transfection, trypsin-EDTA solution (Corning) was used to dissociate the transfected cells at 37 ° C for 2 minutes. Subsequently, 2 ml of warm Dulbecco's modified Eagle medium (DMEM) (Corning) was added, which was supplemented with 10% fetal bovine serum (FBS) (GeminiBio-Products). The resuspended cells were transferred to a 15 ml Falcon tube and centrifuged at room temperature for 5 minutes at 1000 rpm. The medium was then removed and the cells were resuspended in 0.4–1 ml DMEM. The cells were filtered through the 50 μm mesh cap of a CellTrix filter (Sysmex). Cells expressing GFP were sorted by flow cytometry into 5 ml polypropylene round-bottom tubes (Corning) for immediate DNA extraction.

[0181] Isolate the original DNA from the sorted cells. Add proteinase K (20 mg / ml) to DirectPCR lysis reagent (Viagen Biotech Inc.) to a final concentration of 1 mg / ml. 4 – 1×10 5 ) Centrifuge at 12,000 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend the cell pellet in 20–50 μL DirectPCR / Proteinase K solution, incubate at 55°C for >2 hours or until no clumps are observed, incubate at 85°C for 30 minutes, and spin briefly (10 seconds). Use 1–2 μL of DNA for PCR amplification. All PCR primer sequences are described in this article.

[0182] PCR amplicon preparation for deep sequencing. To prepare for deep sequencing, PCR amplicons of approximately 300 bp were amplified using the GoTaq kit (Promega), separated on a 2% agarose gel, and purified using the MinElute Gel Extraction Kit (Qiagen). For each sample, 100 ng of gel-purified PCR product was barcoded using the NexteraFlexPrepHT kit according to the manufacturer's instructions and sequenced using the MiSeq double-end-150 cycle format by the Genome Technology Center Core Facility at the New York University Grossman Medical Center.

[0183] Detection of large deletions. Male DMD-del52 iPSCs were electroporated with vectors expressing Cas9, GFP, and guide RNAs G10 or G9, either alone or in combination with T4-WT or T4-D219A. Electroporated cells were then sorted for GFP 72 hours after electroporation. + Population. Expand sorted cells. Isolate DNA from expanded cells 2 weeks later and perform large deletion assay. Isolate single cells from edited cell pool into 96-well plates 2 weeks after electroporation and perform genotyping 2 weeks later. Store single cells containing an insertion of G at DMD exon 51 or T at DMD exon 53 and expand for subsequent experiments. Edited iPSCs and single clones containing 1-bp insertions are further differentiated into iCMs. Isolate DNA from iCMs and perform large deletion assay.

[0184] Detection of chromosomal translocations. HEK293T cells were co-transfected with vectors expressing Cas9, GFP, and guide RNAs targeting ROS1 and CD74 or PDCD1, TRAC, and TRBC1 / TRBC2, either alone or in combination with T4-WT or T4-D219A. Transfected cells were sorted for GFP 72 hours after transfection. + The sorted cells (1×10 6 ) for DNA extraction. Chromosomal translocations were detected by PCR using primers that specifically recognize the breakpoint junction region of each fusion chromosome. Table 1 summarizes all guide RNAs used.

[0185] Human iPSC maintenance and nucleofection. Human iPSC lines were cultured in Stemflex TM Culture medium (ThermoFisher) and passaged approximately every 3 days (1:8–1:12 split ratio). One hour before nucleofection, iPSCs were treated with 10 μM ROCK inhibitor (Y-27632) and dissociated into single cells using Accutase (Innovative Cell Technologies Inc.). Cells (8 × 105 ) was mixed with 2 μg of vectors expressing Cas9, GFP, and guide RNA, and 2 μg of vectors encoding DNA polymerase. The mixture was electroporated into cells using the P3 Primary Cell 4D-Nucleofector X kit (Lonza) according to the manufacturer's protocol. After nuclear transfection, iPSCs were cultured in StemFlex medium supplemented with CloneR (10×) (StemCell Technologies) and antibiotic-antimycotic (100×) (ThermoFisher). Three days after nuclear transfection, cells expressing GFP were sorted as described above and re-seeded into StemFlex medium. 10 to 15 days after sorting, cells were harvested for DNA isolation.

[0186] Cardiomyocyte differentiation and purification. Human iPSCs (edited iPSC pools or individual clones with 1-bp insertions) were induced to differentiate into cardiomyocytes using the PSC Cardiomyocyte Differentiation Kit (ThermoFisher Scientific) according to the manufacturer's instructions. 15-20 days after the start of differentiation, cells were purified in RPMI-1640 medium lacking glucose and supplemented with B27 (ThermoFisher Scientific). Cells were cultured in this medium for 2-4 days. 40-50 days after the start of differentiation, cardiomyocytes were used for experiments.

[0187] RNA extraction and cDNA synthesis. RNA was extracted from iPSC-derived cardiomyocytes using TRIzol (Catalog 15596026; ThermoFisher Scientific) according to the manufacturer's protocol. cDNA was synthesized using Superscript III First-Strand cDNA Synthesis Kit (ThermoFisher LifeTech) according to the manufacturer's instructions. All RT-PCR primer sequences are described in this article.

[0188] Western blotting. HEK293T cells and cardiomyocytes (iCMs) differentiated from iPSCs were harvested, centrifuged, and lysed with RIPA lysis buffer (Santa Cruz Biotechnology) according to the manufacturer's protocol. The samples were lysed and centrifuged, and the supernatant was incubated at 95°C for 10 minutes in the presence of Laemmli sample buffer (catalog 161-0747; Bio-Rad). Proteins (20 μg per sample) were separated on Mini-PROTEAN TGX 4–15% precast SDS-PAGE gels (Bio-Rad) at 120 V for 1-2 hours and then transferred to PVDF membranes at 250 mA for 1-4 hours. The membranes were probed with anti-HA antibody (catalog number M180-3; MBL) and anti-glyceraldehyde-3-phosphate dehydrogenase antibody (catalog number MAB374; Sigma) or anti-dystrophin (catalog number ab7817; abcam) and anti-vinculin antibody (catalog number V9131; Sigma-Aldrich) overnight at 4° C. The membranes were then washed, probed with goat anti-mouse or goat anti-rabbit IgG H+L-HRP-conjugated secondary antibodies (1:10,000) (Bio-Rad) for 1 hour, and visualized by Western blotting with Luminol reagent (Santa Cruz) according to the manufacturer's protocol.

[0189] PCR amplicon preparation for PacBio sequencing. In order to prepare samples for PacBio sequencing, genomic DNA was extracted from iPSC using the DNeasy blood and tissue kit. The barcode was added to the target region by a two-step PCR reaction. According to the manufacturer's instructions, LATaq DNA polymerase (Takara) was used for the first round of PCR. The first round of PCR used target-specific primers with universal forward and reverse sequences to amplify the 5kb region around the target site. The second round of PCR used universal forward and reverse primers with barcodes to re-amplify and barcode the first round of PCR products. The final barcoded PCR product was sequenced using the SMRTCell (1M v3 LR) platform by the core facility of the Genomic Technology Center of the New York University Grossman Medical Center.

[0190] Bioinformatics analysis

[0191] Deep sequencing. To detect indels in deep sequencing data, unaligned paired-end amplicon deep sequencing reads were used as input to the CRISPResso2 tool to quantify the frequency of editing events (44). The tool was run with default parameters (https: / / github.com / pinellolab / CRISPResso2).

[0192] PacBio sequencing. Raw PacBio data were demultiplexed with corresponding barcodes using SMRTlink software to assign barcoded reads to each sample (smrtlink version: 8.0.0.80529, chemistry package: 8.0.0.778409, parameters: 8.0.0). Demultiplexed data were analyzed using PacBio tools distributed through Bioconda (https: / / github.com / PacificBiosciences / pbbioconda). For DMD exon 51 and 53 gene loci stacking, circular consensus sequences were converted to HiFi calls using the pbccs command and reads supported by at least three full-length subreads were filtered out. The generated fastq files were used as input to a custom python script that filtered reads containing specific 50-bp index sequences in the 5' and 3' regions of each read. The resulting filtered reads were aligned to the reference genome using minimap2 (ax splice --splice-flank=no-u no-G 5000). The genome coverage of the alignment files was calculated using the “bedtools genomecov -d” (v2.27.1) command, and all downstream analyses were performed using custom R scripts (v4.1.1) and visualized using the Gviz1 package (45, 46). For DMD exon 51, the 5’ index sequence was tttttccaaacgtgcttttcaggaaacagtggtctgcttgttgaagtctg (SEQ ID NO: 60), and the 3’ index sequence was aatcctggaccagaggttccattgagctgagatcacaccattgcactcca (SEQ ID NO: 61). For DMD exon 53, the 5' index sequence is ggactatatttttgatttcatgttacaatcactagttttgtggggtcttt (SEQ ID NO: 62) and the 3' index sequence is tgatgtgtattgctgcagattcaatgtaagttcccgatacagataaagat (SEQ ID NO: 63).

[0193] Table 1

[0194]

[0195]

[0196] *Intermediates generated during continuous CasPlus editing

[0197] Table 2. Large deletions generated by Cas9 and CasPlus editing using guide RNAs TS10 or TS9 in male DMD-del52 cells

[0198]

[0199] *Only those circular consensus sequencing (CCS) reads that contained both 5' and 3' index sequences were analyzed

[0200] Table 3. Summary of synthetic sequences and vector information used in this disclosure

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

Claims

1. A DNA polymerase protein, optionally present in a fusion protein comprising a fragment of an MS2 bacteriophage coat protein, wherein the DNA polymerase is selected from: i) a T4 DNA polymerase comprising a mutation of D219, wherein the mutation is optionally a D219A mutation; and ii) RB69 DNA polymerase, said RB69 comprising a mutation of D222, wherein said mutation is optionally D222A.

2. The DNA polymerase protein of claim 1, wherein the DNA polymerase is T4 DNA polymerase and comprises a D219A mutation.

3. The DNA polymerase of claim 1, wherein the DNA polymerase is RB69 DNA polymerase protein and comprises a D222A mutation.

4. The DNA polymerase according to any one of claims 1 to 3, wherein the DNA polymerase protein is present in a fusion protein comprising a fragment of MS2 bacteriophage coat protein.

5. A system for editing a DNA substrate, the system comprising a DNA polymerase protein according to claim 4 and a Cas9 nuclease, the Cas9 nuclease optionally comprising a mutation selected from a mutation at position F916, R919 or Q920, wherein the mutation is optionally selected from F916P, F916del, R919P and Q920P and combinations thereof.

6. The system of claim 5, wherein the DNA polymerase is a T4 DNA polymerase protein and comprises a mutation of D219, and wherein the Cas9 nuclease comprises a mutation selected from the group consisting of F916P, F916del, R920P, and Q920P.

7. The system of claim 6, further comprising at least one guide RNA that directs the system to a specific genomic location and generates an insertion / deletion without the use of a DNA repair template, and wherein the guide RNA optionally comprises a MS2 bacteriophage coat protein binding site.

8. The system of claim 7, wherein the DNA polymerase protein comprises a fragment of MS2 bacteriophage coat protein.

9. The system of claim 5, wherein the DNA polymerase protein is an RB69 DNA polymerase protein comprising a mutation of D222, and wherein the Cas9 nuclease comprises a mutation selected from the group consisting of F916P, F916del, R920P, and Q920P.

10. The system of claim 9, further comprising at least one guide RNA that directs the system to a specific genomic location and generates an insertion / deletion without the use of a DNA repair template, and wherein the guide RNA optionally comprises a MS2 bacteriophage coat protein binding site.

11. The system of claim 10, wherein the DNA polymerase protein comprises a fragment of MS2 bacteriophage coat protein.

12. A method comprising introducing the system according to claim 5 into a eukaryotic cell, wherein the DNA polymerase protein, the Cas9 nuclease and the included guide RNA produce an insertion / deletion at a position in the DNA determined by the sequence of the guide RNA.

13. The method of claim 12, wherein the DNA polymerase is a T4 DNA polymerase protein and comprises a mutation of D219, and wherein the Cas9 nuclease comprises a mutation selected from the group consisting of F916P, F916del, R920P, and Q920P.

14. The method of claim 13, wherein the guide RNA optionally comprises a MS2 bacteriophage coat protein binding site.

15. The method of claim 13, wherein the DNA polymerase protein comprises a fragment of MS2 bacteriophage coat protein.

16. The method of claim 12, wherein the DNA polymerase protein is an RB69 DNA polymerase protein and comprises a mutation of D222, and wherein the Cas9 nuclease comprises a mutation selected from the group consisting of F916P, F916del, R920P, and Q920P.

17. The method of claim 16, wherein the guide RNA optionally comprises a MS2 bacteriophage coat protein binding site.

18. The system of claim 17, wherein the DNA polymerase protein comprises a fragment of MS2 bacteriophage coat protein.

19. The method of claim 12, wherein the indel corrects a mutation in a gene associated with muscular dystrophy or cystic fibrosis.

20. The method of claim 12, wherein the eukaryotic cell is a leukocyte.

21. The method of claim 20, wherein the eukaryotic leukocyte is a T cell.

22. The method of claim 21, wherein the indel is located in one or more of PDCD1, TRBC1, TRBC2 or TRAC.

23. The method of claim 22, wherein the T cells are also modified such that they express a chimeric antigen receptor.

Images