WO2023011638A1 - 融合蛋白以及其使用方法 - Google Patents

融合蛋白以及其使用方法 Download PDF

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WO2023011638A1
WO2023011638A1 PCT/CN2022/110597 CN2022110597W WO2023011638A1 WO 2023011638 A1 WO2023011638 A1 WO 2023011638A1 CN 2022110597 W CN2022110597 W CN 2022110597W WO 2023011638 A1 WO2023011638 A1 WO 2023011638A1
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cells
protein
amino acid
cas9
crispr
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胡家志
尹建行
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北京大学
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Definitions

  • the present invention relates to the field of proteins, in particular to fusion proteins for gene editing.
  • T lymphocytes In addition, current medical and scientific researchers often isolate, activate, ex vivo expand T lymphocytes, and inject them into patients for adoptive T cell immunotherapy (Rosenberg et al., 2008; Laskowski and Rezvani, 2020).
  • T cells can eventually be engineered to accommodate synthetic chimeric antigen receptors (CAR) or neoantigen-specific T cell receptors ( T cell receptor, TCR) to enhance its anti-tumor function (Morgan et al., 2006; Kochenderfer and Rosenberg, 2013; Porter et al., 2011; June and Sadelain, 2018).
  • CAR synthetic chimeric antigen receptors
  • T cell receptor TCR
  • CAR T cells attack by directly recognizing tumor cells using the antigen-binding domain of the antibody, thereby bypassing the requirement of the Major histocompatibility complex (MHC) to present tumor antigens (Kalos and June, 2013; Rafiq et al al., 2020).
  • MHC Major histocompatibility complex
  • Both CAR and TCR T cells have shown great potential in cancer therapy, especially for B-cell malignancies, and hundreds of clinical trials have been approved in addition to several commercially available CAR T therapies (Wei et al., 2020; Manfredi et al., 2020).
  • related work used gene editing to inactivate genes that lead to T cell dysfunction or exhaustion to develop next-generation adoptive T cell immunotherapy (Hendriks et al. , 2020).
  • related work has targeted the TCR ⁇ and ⁇ chain genes TRAC and TRBC at exons in the constant region to reduce mismatches of transduced tumor-specific TCRs in TCR T cells and enhance TCR in CAR T cells.
  • ZFN zinc finger nucleases
  • transcription activator-like effector nucleases Transcription activator-like effector nucleases, TALEN
  • CRISPR-Cpf1 clustered regularly interspaced short palindromic repeats
  • CRISPR-Cas9 generates DNA double-strand breaks (DSBs) at targeted genes to efficiently inactivate multiple genes simultaneously in engineered T cells (Stadtmauer et al., 2020; Liu et al. al., 2016; Ren et al., 2017).
  • DSBs DNA double-strand breaks
  • chromosomal translocations often form between two different target loci (Stadtmauer et al., 2020; Liu et al., 2016; Ren et al., 2017).
  • Chromosomal translocations are estimated to occur with a frequency of 1 in 1,000 to 1 in 100 for induced DSBs or DSBs derived from endogenous physiological activities in the cell (Roukos et al., 2013; Hu et al., 2016; Tubes and Nussenzweig, 2017). Furthermore, chromosomal translocations are frequently observed in T-cell acute lymphoblastic leukemia and more than one-third involve TCR loci (Graux et al., 2006; Cauwelier et al., 2006). Chromosomal translocations generated during gene editing hinder its further application.
  • the inventors found in previous studies that during the manufacture of engineered T cells for adoptive cell therapy, a large number of chromosomal translocations were formed in multiple target sites and their off-target sites. This is due to the fact that the perfect repair product cut by the CRISPR-CAS system can be cut repeatedly, further increasing the cutting frequency. The existence of the above-mentioned chromosomal translocation further poses a huge challenge to the safety of gene editing technology.
  • the inventors creatively found that if the Cas protein is fused with a nuclease (such as an exonuclease) to form a Cas internal and external double nuclease, it can greatly reduce the frequency of perfect repair, thereby greatly reducing the CRISPR - The level of chromosomal translocations caused by the CAS system. That is, fusion proteins obtained by linking a protein with exonuclease activity to an RNA-guided endonuclease such as a Cas protein (either directly or through a linker) can reduce or eliminate cellular modifications (such as CRISPR-Cas9-based Chromosomal translocations or deletions in cell modification).
  • a nuclease such as an exonuclease
  • fusion protein of the present invention was utilized in CRISPR technology for multiple gene editing in chimeric antigen receptor (CAR) T cells, and deleterious TCR-involved chromosomal translocations were almost eliminated before infusion.
  • CRISPR chimeric antigen receptor
  • using the fusion protein of the present invention in CRISPR technology can also inhibit chromosomal translocation and improve the editing efficiency of single gene editing. Therefore, the fusion protein of the present invention is an effective genome editing tool, has higher safety, and can maintain or improve gene editing efficiency at the same time.
  • the invention provides that the fusion protein comprises a first protein and a second protein, wherein
  • the first protein is Cas protein, TALEN or ZFN;
  • the second protein is exonuclease or endonuclease, such as TREX2 (three prime repair exonuclease 2), TREX1 (three prime repair exonuclease 1), APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1), Artemis protein, CtIP (CtBP-interacting protein), Exo1 (exonuclease 1), Mre11 (MRE11 homolog, double strand break repair nuclease), RAD1 (RAD1 checkpoint DNA exonuclease), RAD9 protein, Tp53 (tumor protein p53), WRN protein, exonuclease V, T5 nuclease or T7 exonuclease.
  • TREX2 three prime repair exonuclease 2
  • TREX1 three prime repair exonuclease 1
  • APE1 apurinic/apyrimidinic endodeoxyribonu
  • the Cas protein is a Cas9, Cas12a, Cas12b or Cas12e protein.
  • the first protein and the second protein are linked directly or via a linker.
  • the first protein can be at the C-terminus or N-terminus of the fusion protein.
  • the Cas9 is SpCas9 or SaCas9.
  • Cas12a is AsCas12a and Cas12e is PlmCas12e.
  • the linker is a (G)n, (GGGGS)n, (EAAAK)n, (XP)n or XTEN linker, wherein n is an integer greater than or equal to 1 and X is any amino acid, preferably alanine, Lysine or glutamic acid.
  • the linker is a (G)4S linker.
  • n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, or any integer therebetween.
  • X is any naturally occurring or non-naturally occurring amino acid.
  • the TREX2 protein comprises the amino acid sequence of SEQ ID NO.8 or the amino acid sequence of one or more of R163A, R165A and R167A introduced therein, or at least 85%, 90%, 95%, 96% with them , 97%, 98% or 99% sequence identity of amino acid sequences.
  • the TREX2 protein comprises:
  • the Cas9 protein comprises:
  • the fusion protein comprises
  • Cas12e protein contains:
  • Cas12a protein contains:
  • the invention provides a nucleic acid encoding a fusion protein according to the invention.
  • the invention provides a vector comprising a nucleic acid according to the invention.
  • the invention provides a host cell comprising a vector of the invention.
  • the present invention provides a method for preparing the fusion protein of the present invention, which comprises the following steps:
  • the present invention provides a composition comprising a fusion protein of the present invention and sgRNA.
  • the composition is a ribonucleoprotein complex.
  • the ribonucleoprotein complex is a CRISPR-Cas system.
  • the CRISPR-Cas system is a CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas12b or CRISPR-Cas12e system.
  • the present invention provides a method for genetically modifying a cell, the method comprising the step of introducing the fusion protein, nucleic acid, vector or composition according to the present invention into the cell.
  • the cells are T cells or HSC cells.
  • the cells are TCR T cells, NK cells or CAR-T cells.
  • the present invention provides a method for reducing or eliminating the chromosomal structural variation produced when using CRSPR-Cas for gene editing, which includes introducing the fusion protein, nucleic acid, vector or composition of the present invention into target cells step.
  • the cells are T cells or HSC cells, preferably, wherein the cells are TCR-T cells or CAR-T cells.
  • the present invention provides a gene editing method, which includes the step of introducing the fusion protein, nucleic acid, vector or composition of the present invention into target cells, preferably, wherein the cells are T cells or HSC cells .
  • the cells are TCR-T cells or CAR-T cells.
  • the composition is capable of reducing or eliminating abnormal chromosomal structures during gene editing.
  • the gene is selected from the group consisting of Oct4, MYH7, TRAC, PDCD1, TRBC, UROS, Fzd3, TLX1 (HOX11), TLX3 (HOX11L2), HOXA cluster, HAL1, HAL2, LYL1, BHLHB1, LMO1, LMO2, LCK, NOTCH1, CCND2, SIL/TAL1, CALM/AF10, MLL/ENL, MLL/AF6, MLL/AF10, MLL/AFX1, MLL/AF4, NUP214/ABL1, EML1/ABL1, ETV6(TEL)/ABL1, ETV6(TEL )/JAK2, BCR/ABL1, NUP98/RAP1GDS1, B2M, CCR5 or VEGFA.
  • the abnormal chromosomal structure is a chromosomal translocation and/or a chromosomal deletion.
  • the chromosomal deletion is a deletion of a large segment of a chromosome.
  • the large chromosomal deletion is a 100bp-4Mb chromosomal segmental deletion.
  • the large deletion of a chromosome can be 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb, 5kb, 10kb, 20kb, 30kb, 40kb, 50kb, 100kb, 200kb, 300kb, 500kb , 600kb, 700kb, 800kb, 1Mb, 2Mb, or 3Mb deletion of a large segment of chromosome.
  • gene editing is performed by a CRISPR-Cas system, preferably by a CRISPR-Cas9 system, CRISPR-Cas12a, CRISPR-Cas12b or CRISPR-Cas12e.
  • the invention provides cells that have been modified by the methods of the invention.
  • the cells are T cells or HSC cells.
  • the cells are TCR T cells or CAR-T cells.
  • the invention provides methods of treating disease using the cells of the invention.
  • the invention provides the use of the cells of the invention for the manufacture of a medicament for the treatment of a disease in a subject in need thereof.
  • the disease is a genetic disease such as cancer, autoimmune disease or eye disease.
  • the cells of the invention can be used in cell therapy.
  • the cells are autologous or allogeneic.
  • the present invention provides the use of the fusion protein, nucleic acid, vector or composition of the present invention to reduce or eliminate abnormal chromosome structures in the process of gene editing.
  • the gene is selected from Oct4, MYH7, TRAC, PDCD1, TRBC, UROS, Fzd3, TLX1 (HOX11), TLX3 (HOX11L2), HOXA cluster, HAL1, HAL2, LYL1, BHLHB1, LMO1, LMO2, LCK, NOTCH1, CCND2, SIL/TAL1, CALM/AF10, MLL/ENL, MLL/AF6, MLL/AF10, MLL/AFX1, MLL/AF4, NUP214/ABL1, EML1/ABL1, ETV6(TEL)/ABL1, ETV6(TEL)/ JAK2, BCR/ABL1, NUP98/RAP1GDS1, B2M, CCR5 or VEGFA.
  • the abnormal chromosomal structure is a
  • the chromosomal deletion is a deletion of a chromosomal segment of 100bp-4Mb.
  • the gene editing is performed by a CRISPR-Cas system, preferably by a CRISPR-Cas9 system, CRISPR-Cas12a, CRISPR-Cas12b or CRISPR-Cas12e.
  • a CRISPR-Cas system preferably by a CRISPR-Cas9 system, CRISPR-Cas12a, CRISPR-Cas12b or CRISPR-Cas12e.
  • gene editing can be single gene editing or multiple gene editing.
  • the present invention provides fusion proteins, nucleic acids, vectors or compositions that ensure genome stability during gene editing, improve the genome stability of T cells during CAR-T construction, or prevent CAR-T cells from being transformed. Use in neoplasia. This use is achieved by reducing the abnormal structure of chromosomes.
  • the fusion protein of the present invention can significantly weaken or eliminate large deletions in chromosomes during gene editing;
  • the fusion protein of the present invention can significantly weaken or eliminate chromosomal translocation during gene editing;
  • the fusion protein of the present invention has considerable or even slightly higher gene editing efficiency while achieving the above points 1 and 2;
  • Figure 1 Cas9 mediates chromosomal translocations during multiplex gene editing in human T cells.
  • A Schematic representation of the assessment of chromosomal translocations during multiplex gene editing with Cas9 RNP in human T cells.
  • B PEM-seq detects the editing efficiency of TRAC, TRBC and PDCD1 in human T cells on days 3, 7 and 14 after transfection. The data are from the mean ⁇ SD of three replicates, represented by "circles”, “triangles” and “squares", respectively. Data inspection was performed using a two-tailed t-test, *p ⁇ 0.05 and **p ⁇ 0.01.
  • C Circos plot showing chromosomal translocations detected by PEM-seq between TRAC, TRBC, PDCD1 and TRAC off-target sites in human T cells 3 days after transfection.
  • Clockwise indicates the direction of chromosome arrangement, centromere to telomere. The average value of three replicates is marked on the connection line of chromosomal translocation, and the arrow direction of the connection line indicates the direction of bait and prey.
  • F Gene annotation using Enrichr's KEGG for total chromosomal translocations identified from TRAC, TRBC, and PDCD1 in human T cells 3 days after Cas9 RNP transfection (maayanlab.cloud/Enrichr/). The horizontal axis represents the number of genes in the indicated pathways.
  • G Percentage of Cas9 and corresponding variant enzyme-induced chromosomal translocations detected by PEM-seq in human T cells cloned from TRAC at 3 days after transfection.
  • FIG. 1 CRISPR-Cas9 repeat cleavage enhances off-target bias.
  • Cas9 TRAC in vitro digestion of TRAC on-target sites, off-target sites and chromosomal translocation products (as shown in Figure 2, panel B). Black arrows indicate DNA markers with a size of 400 bp. Cut DNA is indicated by black brackets. D. Percentage of "retargetable” and “nontargetable” chromosomal translocation products in human T cells cloned at the target site in TRAC detected by PEM-seq. Engagement numbers are in parentheses. Off-target site bias was calculated as the ratio of "non-targetable” to "retargetable”. E. Off-target bias of TRAC, TRBC and PDCD1 off-targets detected by PEM-seq in HEK293T cells.
  • G Percentage of off-target chromosomal translocations (top) and off-target bias (bottom) for three off-target sites (OT1 to OT3) of the HBA1 locus in K562 cells. Fold change and off-target bias are indicated on the bars. Off-target sites with little engagement are highlighted with *.
  • Figure 3 Reduction of repeat cutting and chromosomal translocations by fusing TREX2 to Cas9.
  • A Schematic representation of the Cas9-TREX2 fusion protein. Cas9 and TREX2 are linked by a GGGGS linker. R163, R165 and R167 are essential for DNA-binding activity, whereas H188 is essential for the exonuclease activity of TREX2.
  • B PEM-seq detects the editing efficiency of Cas9, Cas9-T2A-TREX2, Cas9X2 and Cas9X2d at the HBA1 locus in HEK293T cells. Gray bars indicate the ratio of deletions and dark bars indicate the ratio of insertions. Deletion rate and total editing efficiency are marked on each bar.
  • C and D are schematic representation of the Cas9-TREX2 fusion protein. Cas9 and TREX2 are linked by a GGGGS linker. R163, R165 and R167 are essential for DNA-binding activity, whereas H188 is essential for the exonuclease activity of TREX2.
  • B PEM-seq detects
  • the percentage of off-target chromosomal translocations is marked in light color, and the percentage of general chromosomal translocations is marked in black.
  • F-I For Cas9, Cas9-T2A-TREX2, Cas9X2 and Cas9X2d PEM-seq libraries in HEK293T cells, editing efficiency (F), deletion rate among editing events (G), percentage of off-target chromosomal translocations (H), general Percentage of chromosomal translocations (I).
  • n 10, the loci used were DNMT1-1, DNMT1-2, EMX1, HBA1, C-MYC1, C-MYC2, CMYC3, RAG1A, RAG1B and RAG1C. Note that for Cas9X2, no off-target chromosomal translocations were detected in several loci. Wilcoxon test, *p ⁇ 0.05, **p ⁇ 0.01, n.s means no significance.
  • (A) In vitro cleavage assay measuring the digestion of 38-mer oligonucleotides by Cas9X2 and Cas9TX in the absence of sgRNA. The indicated amount of protein was incubated with 7.5 nM 38-nt oligonucleotide for the indicated time. NC, BSA served as negative control.
  • B and C PEM-seq detection of Cas9, Cas9X2, Cas9TX editing efficiency (B), off-target chromosomal translocation percentage (C, upper part) and off-target bias (C, lower part) at HBA1 and C-MYC2 in HEK293T cells. As shown in Figure 12, Panel C, the indicated HBA1 off-target site is OT3.
  • FIG. 5 Cas9TX suppresses chromosomal translocations to the level of base editors.
  • A Editing patterns around break sites of BE4max, ABEmax, Cas9 and Cas9TX at the RAG1C locus detected by PEM-seq. Arrows indicate putative fracture sites. Cumulative levels of mutations, deletions and insertions are displayed at nucleotide resolution.
  • B and C For EMX1, C-MYC2, DNMT1-2, RAG1C and BCL11A in HEK293T cells, the editing efficiency of Cas9, Cas9TX, BE4max and ABEmax detected by PEM-seq (B) and the general chromosomal translocation percentage (C ).
  • D Circos diagram showing the distribution of chromosomal translocations of Cas9, Cas9TX, BE4max and ABEmax at RAG1C in HEK293T cells. Red arrows indicate RAG1C target sites. The numbers in the Circos plot show the percentage of general chromosomal translocations.
  • Figure 6 Assessing the effect of Cas9TX on genome stability.
  • A Immunofluorescence of ⁇ H2AX in HEK293T cells treated with etoposide, Cas9 or Cas9TX. The picture is a confocal microscope image taken 24 hours after transfection. Both Cas9 and Cas9TX were expressed with the P2A-mCherry tag. mCherry alone is a negative control.
  • B Statistics of ⁇ H2AX foci in each HEK293T cell treated with etoposide, Cas9 or Cas9TX. Two-tailed t-test, ****p ⁇ 0.00001; n.s, not significant.
  • C Cost of ⁇ H2AX foci in each HEK293T cell treated with etoposide, Cas9 or Cas9TX. Two-tailed t-test, ****p ⁇ 0.00001; n.s, not significant.
  • FIG. 7 Cas9TX abolishes chromosomal translocations in CAR-T cells.
  • A Schematic diagram of Cas9 or Cas9TX RNP preparation of CAR-T cells. Human T cells obtained from human cord blood were activated by anti-CD3/CD28 for 3 days, infected by lentivirus carrying anti-CD19 scFV, and then transfected with Cas9/Cas9TX RNPs. A CAR-T killing assay was performed 3 days after transfection, and chromosomal translocations among TRAC, TRBC, and PDCD1 were analyzed by PEM-seq at 3, 7, and 14 days after transfection. B.
  • T cells infected by lentivirus carrying CAR-CFP detected by FACS Mean ⁇ SD of 3 replicates; ns, not significant.
  • E and F Circos plots representing the chromosomal translocation of Cas9 (E) or Cas9TX (F) between TRAC, TRBC, PDCD1 and TRAC off-target detected by PEM-seq at day 3 after RNP transfection.
  • TRAC off-target sites as described in the legend to Figure 1C.
  • GI Chromosomal translocation induced by Cas9 or Cas9TX and TRAC general chromosomal translocation of TRAC-TRBC (G), TRAC-PDCD1 (H) cloned from TRAC as determined by PEM-seq at 3, 7, and 14 days after transfection Bit (I) ratio. Means of triplicate are shown. Two-tailed t-test, *p ⁇ 0.05, **p ⁇ 0.01.
  • J Evaluation of the killing ability of Cas9 and Cas9TX edited CAR-T cells by CD19 + K562 killing assay.
  • T represents target cells
  • E represents effector cells.
  • FIG. 8 Cas9TX reduces large chromosome segment deletions during multiplex gene editing in CAR-T cell engineering.
  • A Schematic representation of large deletion formation during genome editing. DNA excision, followed by Cas9 cleavage, results in DNA loss resulting in massive DNA deletion.
  • Figure 9 Schematic representation of A. PEM-seq identification of CRISPR-Cas9 editing results.
  • B Schematic showing the PEM-seq library built using two reverse primers (left panel) and the off-target bias detected using the reverse primer PEM-seq library (right panel). Cas9 can persist at the break-end containing sgRNA. Arrows indicate bait primers for PEM-seq.
  • D and E PEM-seq detection of the deletion rate (D) and editing efficiency (E) of Cas9 targeting HBA1, C-MYC1 and C-MYC2 sites in K562 normal cell cycle cells and G1 cells.
  • F PEM-seq detects the percentage of chromosomal translocations at off-target sites of Cas9 targeting C-MYC1 and C-MYC2 in K562 normal cell cycle cells and G1 cells. Fold change is at the top of the bars.
  • Figure 11 A. Off-target bias of Cas9, Cas9-T2A-TREX2, Cas9X2 and Cas9X2d at the HBA1 site in HEK293T cells detected by PEM-seq. The number of identified off-target junctions and the calculated bias are shown at the bottom and the total number of editing events is shown. Red numbers highlight sites with little junction.
  • B Circos plot showing the distribution of genome-wide chromosomal translocations from C-MYC2 clones, as determined by PEM-seq, in HEK293T cells, such as described in Panel E of Figure 3 .
  • n 10, the loci used were DNMT1-1, DNMT1-2, EMX1, HBA1, C-MYC1, C-MYC2, C-MYC3, RAG1A, RAG1B and RAG1C. Note that no off-target junctions were detected in several loci for Cas9X2.
  • Figure 12 A. SDS PAGE showing purified TREX2, TREX2-3R, Cas9X2 and Cas9TX.
  • Figure 13 A. PEM-seq detection of editing results of BE4max, ABEmax, Cas9 and Cas9TX at the RAG1C locus. The percentage of edited products is shown on the right.
  • C Percentages of general chromosomal translocations of Cas9, Cas9TX, BE4max and ABEmax at EMX1, C-MYC2, DNMT1-2, RAG1C and BCL11A detected by PEM-seq in HEK293T cells.
  • Figure 14 A-F. PEM-seq detection, PDCD1-TRAC (A), PDCD1-TRBC (B), TRBC-TRAC (C) of Cas9 and Cas9TX in CAR T cells 3 days, 7 days and 14 days after transfection , TRBC-PDCD1 (D), TRBC general chromosomal translocation (E), PDCD1 general chromosomal translocation (F) chromosomal translocation ratio. Mean ⁇ SD of 3 replicates, means are shown on top of each point. Two-tailed t-test, *p ⁇ 0.05, **p ⁇ 0.01. (G). The percentage of CD19+ and CD19-K562 control cells co-cultured with CAR-T cells at the indicated ratios was detected by FACS 24 hours after mixing. CD19+K562 cells carry BFP, and CD19-K562 cells carry GFP.
  • Figure 15 A model of Cas9TX reducing chromosomal translocations.
  • A. Each round of Cas9 cleavage produces complete rejoining, indels, and chromosomal translocations. Complete repair products can be cut repeatedly by Cas9, with levels of insertions/deletions and chromosomal translocations accumulating in each round of cutting.
  • B. Cas9TX inhibits chromosomal translocations by enhancing indels that are primarily resistant to repeat cleavage, thereby reducing/inhibiting complete rejoining.
  • PEM-seq Primary-extension-mediated sequencing, PEM-seq
  • the inventors traced the sources of various DNA abnormal chromatin repair structures, and finally developed fusion proteins to eliminate chromosomal translocations and large Abnormal chromatin structure such as fragment deletion.
  • the inventor first tested the fusion protein of the present invention in the human 293T cell line, and found that the levels of chromosomal translocation and large fragment deletion were reduced by several times to tens of times.
  • the inventors employed a primer extension-mediated chromosomal translocation sequencing assay to sensitively detect TRAC, TRBC at days 3, 7, and 14 after editing.
  • Chromosomal translocations in targeted T cells at the PDCD1 gene Chromosomal translocations in targeted T cells at the PDCD1 gene.
  • the inventors observed a large number of chromosomal translocations at day 3 post-transfection, and at day 14 these chromosomal translocations remained numerous.
  • the inventors further found that high levels of chromosomal translocations in CRISPR-Cas9-edited T cells were due to repeated cleavage at the target site. Therefore, a new fusion protein was developed that prevents re-cutting and thereby greatly suppresses chromosomal translocations during multiple genome editing in engineered T cells.
  • the fusion protein of the present invention also has enhanced editing ability at many targeted positions compared to wild-type Cas9. It is worth noting that, in addition to playing a role in multi-gene editing, the fusion protein of the present invention can improve editing efficiency when performing single-gene editing and eliminate high-level chromosomal translocation when
  • the PEM-seq (Primer-extension-mediated sequencing, PEM-seq) method is a high-throughput sequencing method for simultaneously determining the editing efficiency and specificity of CRISPR/Cas9, which combines linear amplification-mediated
  • the high-throughput genome-wide translocation sequencing (LAM-HTGTS) and targeted sequencing technology can selectively detect CRISPR/Cas9 off-target sites through translocation capture and evaluate editing efficiency by quantifying Cas9-induced incomplete DSB repair products (Yin , J., Liu, M., Liu, Y., Wu, J., Gan, T., Zhang, W., Li, Y., Zhou, Y., and Hu, J. (2019).
  • PEM-seq has two additional advantages over currently used detection methods: (1) primer extension and random molecular barcode (RMB) in PEM-seq eliminate other methods ( Amplification bias during PCR amplification as used in T7EI, RFLP, TIDE, and targeted sequencing), and (2) PEM-seq to detect small insertions/deletions, large deletions, chromosomal translocations (both CRISPR/Cas9 editing events), while other methods only detect small insertions/deletions.
  • RMB random molecular barcode
  • the PEM-seq method includes primer extension, bridge adapter ligation, PCR amplification, and capture by Illumina sequencing. Specifically, a biotin primer is placed within a specific distance, eg, 200-bp, of the cleavage site, and the biotin primer and sonicated genomic DNA are subjected to repeated annealing and denaturation. The product was purified and heated and quickly cooled on ice for DNA denaturation, and the biotinylated product was purified with streptavidin beads. After washing the DNA-beads, bridge adapter ligation was performed with T4 DNA ligase. Afterwards, nested PCR was performed on the bead-DNA complexes with I5 and I7 sequencing primers. PCR products were recovered by size selection beads, followed by PCT tagging with Illumina P5 and P7 primers. All PEM-seq libraries are sequenced (e.g. 2 ⁇ 150bp Hiseq).
  • gene editing is the targeted modification or modification of the genome of an organism and its transcripts to change the sequence, expression level or function of a target gene or regulatory element.
  • Early gene editing techniques included homing endonucleases, zinc finger endonucleases, and transcriptional activator-like effectors.
  • CRISPR/Cas9 system new technologies represented by the CRISPR/Cas9 system have rapidly expanded the research and application fields of gene editing.
  • Single gene editing refers to gene editing of a single gene of an organism.
  • Multiple gene editing refers to gene editing of multiple genes of an organism at the same time.
  • CRISPR-Cas is a nuclease system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and a CRISPR-binding protein (i.e., Cas protein).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas protein a CRISPR-binding protein
  • the genomic sequence adjacent to the protospacer-adjacent motif (PAM) is cut.
  • CRISPR systems are characterized by elements that facilitate the formation of the CRISPR complex at the site of the target sequence (also known as a protospacer in endogenous CRISPR systems).
  • CRISPR refers to clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats), which are the immune system of many prokaryotes.
  • a guide RNA generally includes a guide sequence and a backbone sequence, and these two sequences can be in the same molecule or in different molecules.
  • the function of the guide RNA is to guide the Cas protein to cut the DNA site complementary to the guide sequence, that is, the target sequence.
  • a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target sequence to hybridize to the target sequence and direct the specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between the guide sequence and its corresponding target sequence is about or more than about 50% or more.
  • leader sequences are about or more than about 12 nucleotides in length.
  • Guide RNA includes single-stranded guide RNA (sgRNA) and double-stranded guide RNA composed of crRNA and tracrRNA.
  • the guide RNA may be a single-stranded guide RNA (sgRNA).
  • target sequence refers to the target sequence against which a complementary guide sequence is designed, wherein hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Perfect complementarity is not required, provided that sufficient complementarity is present to cause hybridization and facilitate formation of the CRISPR complex.
  • the target polynucleotide of the CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide that resides in the nucleus of a eukaryotic cell.
  • a target polynucleotide can be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or dummy DNA).
  • this target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex.
  • the target sequence may be a gene selected from the group consisting of Oct4, MYH7, TRAC, PDCD1, TRBC, UROS, Fzd3, TLX1 (HOX11), TLX3 (HOX11L2), HOXA cluster, HAL1, HAL2, LYL1, BHLHB1, LMO1, LMO2, LCK, NOTCH1, CCND2, SIL/TAL1, CALM/AF10, MLL/ENL, MLL/AF6, MLL/AF10, MLL/AFX1, MLL/AF4, NUP214/ABL1, EML1/ABL1, ETV6(TEL) /ABL1, ETV6(TEL)/JAK2, BCR/ABL1, NUP98/RAP1GDS1, B2M, CCR5, or VEGFA.
  • Cas protein refers to a CRISPR-associated protein.
  • Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1 , Csf2, Csf3, Csf4, and the newly discovered Cas12e, its homologues, or its modified forms.
  • Cas9 protein also known as Csn1
  • Csn1 is a protein involved in both crRNA biosynthesis and destruction of invading DNA. Cas9 has been described in different bacterial species such as S. thermophiles, Listeria innocua and S. pyogenes.
  • the Cas9 protein contains two predicted nuclease domains, an HNH (McrA-like) nuclease domain located in the middle of the protein and a split RuvC-like nuclease domain (RNAase H fold).
  • a Cas9 variant may be a Cas9 endonuclease that does not naturally occur in nature and is obtained by protein engineering or by random mutagenesis.
  • Cas9 variants can be obtained by mutation, ie deletion or insertion or substitution of at least one residue in the amino acid sequence of the Streptococcus pyogenes Cas9 endonuclease.
  • the Cas9 protein is a S. pneumoniae, S. pyogenes, or S. thermophilus Cas9, and may include mutated Cas9s derived from these organisms. These Cas9s are known.
  • the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2
  • the amino acid sequence of the Neisseria meningitides Cas9 protein can be found in the UniProt database under the accession number A1IQ68
  • the Streptococcus thermophilus ( The amino acid sequence of the Streptococcus thermophilus) Cas9 protein can be found in the UniProt database numbering Q03LF7
  • the amino acid sequence of the Staphylococcus aureus (Staphylococcus aureus) Cas9 protein can be found in the UniProt database numbering J7RUA5.
  • the Cas9 protein may comprise the amino acid sequence of SEQ ID NO: 2 or may comprise the sequence of addition, deletion, substitution or insertion of the amino acid sequence of SEQ ID NO. 2, as long as the resulting sequence has the natural function of the Cas9 protein.
  • Cas12a protein is a single-stranded RNA-guided endonuclease lacking a small trans-coding RNA.
  • the Cas12a protein uses a T-rich protospacer-adjacent motif comprising a 2-6 base pair DNA sequence followed by a DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
  • Cas12a protein recognizes T-rich PAM, TTTN, but this sequence is on the 5' side of the primer.
  • the Cas12a protein cleaves DNA through staggered DNA double-strand breaks.
  • the Cas12a protein has recently been used in optional genome editing efforts as a molecular scissors useful in gene editing.
  • Cas12a Compared with the Cas9 system, the editing efficiency of Cas12a is comparable to that of Cas9, and lower than that of Cas9 in some targets.
  • the off-target rate of Cas12a is extremely low.
  • Cas12a is a safe gene editing tool.
  • Cas12a forms a sticky end after cleavage, while Cas9 forms a blunt end. Studies have shown that the sticky end after Cas12a cleavage is more prone to homologous recombination repair than the blunt end of Cas9, which is also the site-specific insertion of genes. and fixes provide better tools. In terms of guide RNA processing, Cas12a has obvious advantages.
  • Cas12a itself can complete the processing of the premise RNA, while the Cas9 system requires the processing of RNaseIII, which greatly promotes the application of Cas12a in multiple gene editing.
  • Cas12a recognizes 5'-TTTN-3' or 5'-KYTV-3'
  • Cas9 recognizes 5'-NGG-3'.
  • the Cas protein may be Cas12a protein, such as AsCas12a protein.
  • the Cas12a protein can comprise the amino acid sequence of SEQ ID NO.91 or be encoded by the nucleic acid comprising SEQ ID NO.92.
  • Cas12e protein is a Cas protein identified by metagenomic analysis of groundwater bacteria and characterized as an RNA-guided DNA nuclease (Liu, J.J. et al. Nature 566, 218–223 (2019). It recognizes 5 '-TTCN PAM, and enables plasmid interference in E. coli when presenting sgRNA (covalently linked crRNA-tracrRNA).
  • Cas12e It bears no similarity to other reported Cas endonucleases except for the RuvC domain located at the C-terminus
  • the above features of Cas12e correlate with those of Cas12; however, the size of Cas12e (about 980 aa) is smaller than the size of Cas12 reported (about 1200 aa).
  • the Liu et al. nt guide fragments and recognize TTCN PAM elements, resulting in cleavage of dsDNA targets with 10-nt staggered ends. It has been reported that Cas12 exhibits PAM-independent nonspecific ssDNA trans-cleavage activity when triggered by binding to crRNA-guided complementary ssDNA. Liu et al. (Burstein, D. et al.
  • Cas albumen can be Cas12e albumen, for example PlmCas12e albumen.Cas12e albumen can comprise the aminoacid sequence shown in SEQ ID NO.89 or can with the nucleic acid coding comprising SEQ ID NO.90.
  • TREX2 protein refers to a protein that has 3' to 5' exonuclease activity and eliminates mismatched, modified, fragmented and normal nucleotides to produce proteins suitable for the subsequent steps of the DNA metabolic pathway. 3' end of the protein. TREX2 plays a role in DNA replication, repair and recombination.
  • the Trex2 protein may comprise the amino acid sequence of SEQ ID NO.1 or may comprise a sequence in which the amino acid sequence of SEQ ID NO.1 has been added, deleted, substituted or inserted, as long as the resulting sequence has the Trex2 protein described herein
  • the Trex2 protein retains amino acid residues 163A, 165A and 167A that eliminate DNA binding activity, or retains amino acid residues that retain its enzymatic activity.
  • a fusion protein can be generated by directly coupling Cas protein, such as Cas9, with a mutant of TREX2 (containing one or more of R163A, R165A and R167A) or coupling with a linker.
  • the Trex2 protein may be wild-type Trex2 protein (SEQ ID NO. 8) or a variant into which one or more of R163A, R165A and R167A is introduced.
  • variable refers to a molecule obtained by mutating or substituting at least one residue in the amino acid sequence of a parent molecule, which molecule retains the functional characteristics of the parent molecule.
  • TALE nuclease or "TALEN” means a fusion protein consisting of a nucleic acid binding domain typically derived from a transcription activator-like effector (TALE) and a nuclease catalytic domain that cleaves a nucleic acid target sequence.
  • the catalytic domain is preferably a nuclease domain, more preferably a domain having endonuclease activity, eg I-TevI, ColE7, NucA and Fok-1.
  • TALE domains may be fused to meganucleases, such as I-Crel and I-OnuI or functional variants thereof.
  • the nuclease is a monomeric TALE nuclease.
  • Monomeric TALE nucleases are TALE nucleases that do not require dimerization for specific recognition and cleavage, such as fusions of engineered TAL repeats and the catalytic domain of I-TevI described in WO2012138927.
  • Transcriptional activators such as effectors (TALEs), proteins from the bacterial species Xanthomonas, contain multiple repeats each containing at positions 12 and 13 specific for each nucleotide in the nucleic acid targeting sequence base two residues (RVD).
  • Binding domains with similar modular base-to-base nucleic acid binding properties could also be derived from new modular proteins recently discovered in different bacterial species.
  • the new modular proteins have the advantage of showing more sequence variability than TAL repeats.
  • the RVDs associated with recognition of different nucleotides are: HD for recognition of C, NG for recognition of T, NI for recognition of A, NN for recognition of G or A, NS for recognition of A, C, G or T, HG, IG that recognizes T, NK that recognizes G, HA that recognizes C, ND that recognizes C, HI that recognizes C, HN that recognizes G, NA that recognizes G, SN that recognizes G or A, YG that recognizes T, recognizes TL for A, VT for A or G and SW for A.
  • TALE nucleases have been described and used to stimulate gene targeting and gene modification (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011).
  • Custom TAL nucleases are commercially available under the tradename TALENTTM (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).
  • the first protein may be any suitable TALEN.
  • a "zinc finger nuclease” or “ZFN” consists of a DNA recognition domain and a non-specific endonuclease.
  • the DNA recognition domain is composed of a series of Cys2-His2 zinc finger proteins in series, generally 3 to 4. Each zinc finger protein recognizes and binds a specific triplet base.
  • Zinc finger proteins originate from the family of transcriptional regulators and are widely present in eukaryotes from yeast to humans, forming alpha-beta-beta secondary structures. Among them, the 16 amino acid residues of the alpha helix determine the DNA binding specificity of the zinc finger, and the skeleton structure is conserved.
  • New DNA-binding specificities can be obtained by introducing sequence changes to the amino acids that determine DNA-binding specificity.
  • the published highly specific zinc finger proteins screened from nature and artificially mutated can recognize all GNNs and ANNs and some CNNs and TNN triplets. Multiple zinc finger proteins can be connected in series to form a zinc finger protein group to recognize a specific base sequence, which has strong specificity and plasticity, and is suitable for designing ZFNs.
  • the non-specific endonuclease associated with the zinc finger protein group comes from the DNA cleavage domain consisting of 96 amino acid residues at the C-terminus of FokI (Kim et al., 1996).
  • FokI is a restriction endonuclease from Flavobacterium seabed, which has enzyme cutting activity only in the dimer state (Kim et al., 1994).
  • Each FokI monomer is connected with a zinc finger protein group to form a ZFN, which recognizes a specific site.
  • the two recognition sites are at an appropriate distance (6-8bp)
  • the two monomer ZFNs interact to produce enzyme cutting function , so as to achieve the purpose of DNA shearing.
  • the first protein may be any suitable ZFN.
  • linker refers to one or more amino acid residues that join two protein parts.
  • the joint may be a flexible joint.
  • the linker is (G)n, (GGGGS)n, (EAAAK)n, (XP)n or XTEN linker, wherein n is an integer greater than or equal to 1, X is any amino acid, preferably alanine, lysine acid or glutamic acid.
  • the linker is a (G)4S linker.
  • n can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, or any integer therebetween.
  • fusion protein refers to a protein formed by linking two or more protein parts, wherein each of the protein parts can perform their functions.
  • the protein parts may be directly connected or may be connected through a linker, as long as the protein parts are each able to perform their functions.
  • a fusion protein of the invention comprises a first protein and a second protein.
  • the first protein is Cas protein, TALEN or ZFN
  • the second protein is exonuclease or endonuclease.
  • the present invention provides fusion protein Cas9TX, which is the first gene editing enzyme mainly used in the field of gene editing to reduce the abnormal chromatin repair structure in the gene editing process.
  • the gene editing enzyme Cas9TX was generated by coupling Cas9 to mutants of TREX2 (R163A, R165A, and R167A) with a (G)4S linker.
  • Cas9TX ensures the genome stability during the gene editing process, reduces the generation of abnormal chromosomal structures and improves the genome stability of T cells during the CAR-T construction process, preventing the transformed CAR-T cells from undergoing tumorigenesis.
  • the fusion protein of the present invention uses a mutant of the endogenous Trex2 protein of the cell, which is safe in use. It is the first gene editing enzyme in the field of gene editing that is mainly used to reduce the abnormal chromatin repair structure in the process of gene editing .
  • the fusion protein comprises the amino acid sequence of SEQ ID NO.3 or may comprise the sequence of addition, deletion, substitution or insertion of the amino acid sequence of SEQ ID NO.3.
  • chromosomal structural variation generally refers to the abnormal connection of segments of chromosomal material in a manner that is absent in wild-type or normal cells.
  • chromosomal structural variations include chromosomal deletions, amplifications, inversions, chromosomal translocations, and the like.
  • structural chromosomal variation may refer to chromosomal deletion and/or chromosomal translocation.
  • chromosomal translocation generally refers to the equal or non-equivalent exchange of chromosomal material between two chromosomes that are the same or different.
  • chromosomal deletion or “chromosomal segment deletion” refers to deletion of a chromosome segment ranging from 100 bp to 4 Mb on a chromosome.
  • Large fragment deletions of chromosomes can be 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb, 5kb, 10kb, 20kb, 30kb, 40kb, 50kb, 100kb, 200kb, 300kb, 500kb, 600kb, 700kb, 80 , 1 Mb, 2 Mb, or 3 Mb of a large segment of chromosome deletion.
  • adoptive cell transfer is the transfer of cells into a patient. These cells may have come from the patient himself and subsequently changed before being transferred back (autologous transfer), or they may have come from another individual. The cells are most commonly derived from the immune system with the goal of transferring improved immune function and properties along with the cells back to the patient. Transfer of autologous or patient-derived cells can minimize graft-versus-host disease (GVHD) or tissue or organ rejection.
  • GVHD graft-versus-host disease
  • chimeric antigen receptor refers to a genetically engineered receptor that can be used to graft one or more antigen-binding moieties to immune effector cells, such as T cells. Some CARs are also called “artificial T cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.”
  • a CAR may comprise an extracellular antigen-binding domain specific for one or more antigens, such as a tumor antigen, a transmembrane domain, and an intracellular signaling domain for T cells and/or other receptors.
  • Chimeric antigen receptor-modified immune cells use genetic engineering to modify immune cells to express exogenous CAR genes.
  • the CAR gene mainly includes an extracellular recognition domain and an intracellular signal transduction domain: the former is used to recognize target cell surface-specific molecules (such as tumor surface-specific molecules), and the latter is used to initiate immune cell responses after recognizing tumor surface molecules , to play a cytotoxic effect.
  • Chimeric antigen receptors are mainly carried by T-cells, and "CAR-T” refers to T cells expressing CAR.
  • T cell receptor refers to the molecule present on the surface of T cells that is responsible for recognizing antigens displayed on the surface of antigen presenting cells (APCs). Each T cell expresses a unique TCR, generated by a random assortment of genes, ensuring that the T cell can respond to virtually any infection. TCRs are also able to recognize tumor-specific proteins (antigens) from inside the cell. When the tumor-specific protein (namely LMP2) is broken down into fragments, it appears on the cell surface along with the major histocompatibility complex (MHC). MHC class I molecules present peptide antigens derived from proteins inside cells. TCRs can be engineered to recognize tumor-specific protein fragment/MHC combinations.
  • APCs antigen presenting cells
  • TCR The structural formula of TCR is composed of two different protein chains including ⁇ chain and ⁇ chain.
  • Heterodimeric TCR proteins typically consist of highly variable ⁇ and ⁇ chains expressed as part of a complex with an invariant CD3 chain molecule.
  • the variable domains of both the alpha and beta chains of a TCR have three hypervariable or complementarity determining regions (CDRs), respectively.
  • TCR T cell refers to a T cell or population of T cells that have been selected, isolated and/or identified by molecular biological methods to express a T cell receptor activated in response to an antigen of interest.
  • the T cell population may comprise peripheral blood mononuclear cells (PBMC).
  • PBMCs may contain lymphocytes (T cells, B cells, NK cells), monocytes and granulocytes (neutrophils, basophils and eosinophils). In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes and a small percentage of dendritic cells.
  • the TCR can be modified on any cell comprising a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and ⁇ T cells.
  • proteins can be appropriately modified to obtain protein variants.
  • one or more, preferably 2, 3, 4 or 5 amino acid sequences can be substituted, added, deleted or inserted into the amino acid sequence of each protein of the present invention, such as the amino acid sequence shown in SEQ ID NO: 1, 2 or 3 Amino acid sequence of amino acid residues.
  • Amino acid addition refers to the addition of amino acids at the C-terminal or N-terminal of an amino acid sequence, such as SEQ ID NO: 1 or 2.
  • Amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as a sequence of SEQ ID NO: 1 or 2, by other amino acid residues.
  • Amino acid insertion refers to the insertion of amino acid residues at appropriate positions in an amino acid sequence such as SEQ ID NO: 1 or 2, and the inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other.
  • Amino acid deletion refers to the deletion of 1, 2 or 3 or more amino acids from the amino acid sequence, such as the sequence of SEQ ID NO: 1 or 2.
  • the substitution may be a conservative amino acid substitution, which means that compared with the amino acid sequence of SEQ ID NO: 1 or 2, there are 3, more preferably 2 amino acids or 1 amino acid are replaced by amino acids with similar or similar properties to form peptides.
  • conservative variant peptides can be produced by amino acid substitutions according to Table 1.
  • conservative substitutions may be defined in terms of substitutions within amino acid classes as reflected in one or more of the following three tables:
  • Protein variants of the invention can be characterized by sequence identity.
  • sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as described in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably implemented in the Needle program above version 5.0.0.
  • the parameters used were a gap opening penalty of 10, a gap extension penalty of 0.5 and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained with the -nobrief option) is used as percent identity and is calculated as follows:
  • a particular protein encompasses the wild-type protein or a protein variant having amino acid sequence identity thereto.
  • Amino acid sequence identity may be at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence.
  • the TREX2 protein covers the amino acid sequence of the wild-type protein of SEQ ID NO.8 or the amino acid sequence of one or more of R163A, R165A and R167A introduced therein, or at least 85%, 90%, 95%, 96% with them , 97%, 98% or 99% sequence identity of amino acid sequences.
  • Intracellular delivery of a protein or composition of the invention can be contemplated by any method known in the art.
  • Non-limiting examples include viral transduction, electroporation transfection, liposome delivery, polymeric carriers, chemical carriers, lipoplexes, polymeric complexes, dendrimers, nanoparticles, emulsions, natural endocytosis or Phagocytosis pathway, cell penetrating peptides, microinjection, microneedle delivery, particle bombardment, etc.
  • a preferred embodiment is electroporation transfection
  • electroporation instruments include: Neon transfection system (Thermo Fisher Scientific), Gemini instrument and AgilePulse/CytoPulse instrument (BTX-Harvard apparatus), 4D - Nucleofector system, Amaxa Nucleofector II, Nucleofector 2b instrument (Lonza), CTX-1500A instrument (Celetrix), MaxCyte GT or VLX instrument (MaxCyte), Gene Pulser Xcell (Biorad).
  • the pulse duration, intensity, interval between pulses, and number of pulses can be modified to achieve optimal conditions for high transfection efficiency and low mortality.
  • the main transfected cell types are human primary T cells, lymphocytes, and peripheral blood mononuclear cells. In theory, most cell types can use the electroporation transfection method to deliver the vector into the cells .
  • the CRISPR-Cas system of the present invention can be used to prepare cells expressing chimeric antigen receptors (CAR) or T cell receptors.
  • the cells to be modified according to the invention may be any suitable T cells.
  • T cells can be inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T cells or helper T lymphocytes.
  • T cells are cytotoxic T lymphocytes.
  • the T cells are selected from CD4+ T lymphocytes and CD8+ T lymphocytes. They can be extracted from blood or derived from stem cells.
  • Stem cells may be adult stem cells, embryonic stem cells, more specifically non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.
  • Typical human cells are CD34+ cells.
  • the T cells to be modified according to the invention are human T cells.
  • the source of cells Prior to expanding and genetically modifying the cells of the invention, the source of cells can be obtained from a subject, such as a patient, by a variety of non-limiting methods.
  • T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available and known to those of skill in the art may be used.
  • cells may be derived from healthy donors or from patients diagnosed with disease.
  • the cell is part of a mixed population of cells exhibiting different phenotypic properties.
  • the present invention also provides encoding nucleic acids of TREX2 protein, Cas9 protein and fusion protein.
  • nucleic acids can be used to express these proteins.
  • various proteins mentioned herein can be expressed in recombinant cells.
  • the type of cell is not limited, for example, the cell may be eukaryotic or prokaryotic.
  • Eukaryotic cells may be fungal cells, such as yeast cells, or insect cells or mammalian cells, such as mouse cells.
  • Prokaryotic cells may be bacterial cells, such as E. coli cells.
  • the nucleic acid of the protein can be codon-optimized depending on the host cell used.
  • the nucleic acid can be cloned into an appropriate expression vector, which is then introduced into host cells for expression.
  • the type of expression vector is not limited and is well known to those skilled in the art.
  • Human primary T cells were obtained from human cord blood using a negative selection kit (STEMCELL Technologies, catalog #19051). T cells were cultured at 37°C and 5% CO in RPMI 1640 (Corning ) containing 30 units/ml recombinant human interleukin 2 (IL-2, Gibco, catalog number PHC0021), glutamine (Corning), 15% fetal bovine serum (FBS, Excell Bio), and penicillin/streptomycin (Corning). T cells were activated with human anti-CD3/28 beads at a 1:1 ratio for 3 days prior to transfection of Cas9 RNP (Thermo Fisher, Cat# 11161D).
  • HEK293T cells cultured in 10 cm dishes were co-transfected with 5 ⁇ g pMD2.G (Addgene 12259), 10 ⁇ g psPAX2 (Addgene 12260) and 20 ⁇ g anti-CD19 scFV 4-1BB plasmids using PEI (Sigma, P3143).
  • the supernatant was filtered with a 0.45 ⁇ m filter and concentrated using a Beckman Optima L8-80XP at 70,000 g for 2 hours at 4 °C.
  • Activated human T cells (1 day after stimulation) were infected with lentivirus at 500 g for 2 h with 8 ⁇ g/mL polybrene (Sigma, TR-1003).
  • Cas9, Cas9 high-fidelity variants Cas9X2 and Cas9TX were expressed using the pET28a backbone (Addgene 53135).
  • TREX2 and TREX2-3R mutants were expressed using pDB-His-MBP (Addgene 123365).
  • the pDB-His-MBP-TREX2 plasmid was transfected into E. coli BL21(DE3) Rosetta cells. This method refers to a previous method (Mazur and Perrino, 2001), except that MBP cleavage was performed using TEV instead of Genenase.
  • the pET28a plasmid was transformed into E. coli BL21(DE3) Rosetta cells and the expression was induced by IPTG (Amresco, 0487).
  • Cells were lysed by sonication in lysis buffer (20 mM HEPES, pH 7.5, 10% glycerol, 0.1% Triton X-100) containing 1 mM PMSF, and cell debris was removed by centrifugation at 20,000 g for 1 hour.
  • the supernatant was loaded onto a HisTrap HP column (GE Healthcare) and eluted with a gradient of imidazole from 0 to 300 mM in lysis buffer. Fractions were then gel filtered on a Superdex200 column (GE Healthcare). Finally, the purified protein was quantified using BSA protein standards and stored in lysis buffer at -80°C until use.
  • GTGTCACAAAGTAAGGATTCTG and CTAGTCTTGTCTGCTACCTGGATC were used for the amplification of the TRAC-TRBC chromosomal translocation
  • GTGTCACAAAGTAAGGATTCTG and GCACCCTCCCTTCAACCTGACCTGGGAC were used for the amplification of the TRAC-PDCD1 chromosomal translocation
  • CTAGTCTTGTCTGCTACCTGGATC and GCACCCTCCCTTCAACCTGACCTGGGAC were used for the amplification of the TRBC-PDCD1 chromosomal translocation .
  • Recover the PCR product and use TTCTGATGTGTATATCACAG and CTAGTCTTGTCTGCTACCTGGATC for the second round of PCR for the amplification of TRAC-TRBC chromosomal translocation, and use TTCTGATGTGTATATCACAG and GAGAAGGCGGCACTCTGGTG for the second round of PCR for the amplification of TRAC-PDCD1 chromosomal translocation.
  • Amplification of translocations A second round of PCR was performed using CTAGTCTTGTCTGCTACCTGGATC and GCTCACCTCCGCCTGAGCAG.
  • PEM-seq libraries were prepared as previously described (Yin et al., 2019).
  • a new chromosomal translocation filter module to filter false chromosomal translocation junctions, including junctions with the same random molecular barcode (RMB) and Identical junction sequences with highly similar RMB ( ⁇ 2 mismatches) (Liu et al., 2021).
  • RMB random molecular barcode
  • PEM-seq can identify genome editing artifacts: complete rejoining, indels, chromosomal translocations, and other chromosomal abnormalities. The ratio of indels to total recognition products was defined as editing efficiency.
  • Indels were defined as deletions ( ⁇ 100bp) and insertions ( ⁇ 20bp).
  • editing efficiency was calculated by counting all products recognized by CRISPResso (>0.2%).
  • the editing frequency of the primary cytosine or adenine was used as the "desired" editing efficiency for BE4max or ABEmax, normalized relative to the same editing efficiency for Cas9 and Cas9TX in panel E of Figure 13 .
  • Chromosomal translocation hotspots with high sequence similarity ( ⁇ 8nt mismatches, considering both sgRNA and PAM sequences) to the target site and with junction at the putative Cas9 cleavage site were considered off-target sites.
  • Overall chromosomal translocations were calculated by excluding junctions within ⁇ 20 kb around target sites and ⁇ 100 bp around off-target sites.
  • Table 4-1 Primer sequences used for PEM-seq library construction The following two sequences are identical and need to be confirmed.
  • sgRNAs fused to scaffold RNAs were transcribed in vitro by using the T7 High Efficiency Transcription Kit (TransGen Biotech). Cas9 at a concentration of 100 nM and 300 nM RNA were included in each reaction. Digest DNA fragments for 2 hours at 37°C under the following conditions: 20mM HEPES (pH 7.5), 5% glycerol, 100mM KCl, 1mM dithiothreitol, 10mM MgCl2 and 0.5mM EDTA.
  • the primers in Table 4 were used to amplify both the on-target and off-target sites in Cas9:TRAC. Construction of the target site in TRAC linked to both ends of the TRAC off-target (retargetable and non-targetable chromosomal translocation products) was generated by overlap PCR.
  • TRAC-F GTGTACCAGCTGAGAGACTC
  • OT-DN-R GGCATAGCCAATCCATTCAGTGATC
  • OT-UP-F TTTAGCATTTACTCAAAAAGTCCACAATCCA
  • TRAC-R TGGTGGCAATGGATAAGGCC
  • OT-DN-F AACTGTGCTAGACATGAGGTACTTCATGTCTTGCATCTGGGTCA
  • OT-UP-R AACTGTGCTAGACATGAGGTCTAAGGAGATCATTTTGGAGTTTTAAGATC
  • K562 cells were treated with 5 ⁇ M palbociclib (PD-0332991) HCl (Selleck, S1116) for 36 hours, then transfected with Cas9 plasmid and re-cultured in 5 ⁇ M palbociclib, after which the cells were harvested.
  • cells were labeled with 50 ⁇ M BrdU for 60 min, fixed with paraformaldehyde (PFA) for 60 min at 4°C, and then incubated with anti-BrdU (100x, BD) for 40 min. Cells were then stained with 7-AAD (25Ox, BD) for 20 minutes and analyzed by FACS.
  • sgRNAs used are listed in Table 5. Construct Cas9, Cas9X2, Cas9-T2A-TREX2, Cas9X2d, Cas9TX, BE4max (a gift from Dr. Chengqi Yi), ABEmax (a gift from Dr. Chengqi Yi) to the pX330 backbone (Addgene 42230) with mCherry driven by P2A-mCherry or CMV middle. sgRNA expression cloned into a different pX330 backbone with CMV-driven GFP instead of Cas9. TREX2-H188A and TREX2-3R mutants were obtained by overlap PCR and confirmed by Sanger sequencing.
  • loci sgRNA sequence RAG1A GCCTCTTTCCCCACCCACCTT GGG RAG1B GACTTGTTTTCATTGTTCTC AGG RAG1C GCACCTAACATGATATATTA AGG DNMT1 TTCCCGGCAGATGTTTACCT TGG DNMT2 CCCTGCAGTTCCCTAACTGA GGG
  • HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Corning) containing glutamine (Corning), 10% fetal bovine serum (FBS, Excell Bio), and penicillin/streptomycin at 37°C, 5% CO 2 Element (Corning).
  • K562 cells were cultured in RPMI 1640 (Corning) containing glutamine, 15% FBS and penicillin/streptomycin (Corning) at 37°C, 5% CO2.
  • a library of HEK293T cells was prepared by co-transfecting 3 ⁇ g of Cas9 plasmid and 3 ⁇ g of sgRNA plasmid with PEI (Sigma) in a 6-cm dish.
  • Cas9 plasmid (1 ⁇ g/1 million cells) and GFP plasmid (1 ⁇ g/1 million cells) were co-introduced into K562 cells by using 4D-nucleofector with FF120 program in SF buffer (Lonza). All sample cells were collected by FACS sorting with mCherry and/or GFP.
  • the reaction assay (10 ⁇ L) contained 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM DTT, 100 ⁇ g/ml BSA, 7.5 nM 38-mer oligonucleotide (Genewiz) and TREX2 protein or Cas9X2. Incubate for indicated times at room temperature. The reaction was terminated by adding 30 ⁇ L of ethanol and dried under vacuum. The pellet was resuspended in 6 ⁇ L of 1 ⁇ loading buffer, denatured at 95 °C for 5 min, and then separated on a 15% denaturing polyacrylamide gel.
  • HEK293T cells were cultured on glass slides in 6-well dishes. After 24 hours of Cas9/Cas9TX transfection or 10 ⁇ M etoposide (Sigma, S1225) treatment, cells on slides were fixed in 4% PFA for 10 minutes at room temperature, followed by PBS washing. Cells were then permeabilized with 0.5% TritonX-100 for 15 minutes. Cells were blocked with 3% FBS for 60 min prior to primary antibody staining. Cells were then incubated with anti- ⁇ H2A.X (phospho-S139) (Abcam, ab2893) diluted 1:500 for 1 hour at room temperature or overnight at 4°C, and then washed with 0.2% Tween.
  • Cas9/Cas9TX transfection or 10 ⁇ M etoposide (Sigma, S1225) treatment cells on slides were fixed in 4% PFA for 10 minutes at room temperature, followed by PBS washing. Cells were then permeabilized with 0.5% TritonX-100 for 15 minutes. Cells were
  • K562 cells with CD19 and BFP expression or K562 cells with FITC (but no CD19) were co-cultured with Cas9/Cas9TX edited CAR-T cells at E:T ratios of 1:1, 5:1, 10:1 for 24 Hour. The killing efficiency was then detected by FACS and analyzed by FlowJo 10.4.
  • Example 1 Extensive chromosomal translocations observed in engineered T cells
  • the primer extension-mediated translocation sequencing method was used in this example to capture chromosomal translocations, and named in this study as PEM-seq.
  • this example places a bait primer on one of the broken ends of the target site to generate a PEM-seq library that can accurately quantify insertions/deletions (indels) between the two broken ends of the target site and Chromosomal translocations between bait break ends and other DSB ends ( Figure 9, panel A; Yin et al., 2019; Liu et al., 2021).
  • the ratio of indels to total sequencing reads was defined as editing efficiency, while percent chromosomal translocations were calculated as the ratio of chromosomal translocations to indels plus chromosomal translocations.
  • T cells were enriched from human umbilical cord blood and activated by anti-CD3/CD28 for 3 days, and then TRAC, TRBC and PDCD1 genes were treated with CRISPR-Cas9, as in clinical protocol NCT03399448 (Stadtmauer et al., 2020) Used.
  • Cas9 protein was purified and mixed with three sgRNAs and delivered as ribonucleoprotein complexes (RNPs); T cells were monitored for editing efficiency and chromosomal translocation 3, 7, or 14 days after transfection ( Figure 1, panels A and Panel B of Figure 9).
  • CRISPR-Cas9 induced substantial editing results in T cells.
  • the editing efficiency of TRAC reached about 51.9%, that of TRAC reached about 44.6%, and that of PDCD1 reached 47.8% ( Figure 1 Figure B and Table 6).
  • the guide RNA (sgRNA) of TRBC has two real target sites with an interval of about 9.4kb between each other in the same TRBC gene, and they are combined for analysis in this embodiment.
  • the percentage of cells containing edited products became lower and lower, which may be due to growth retardation of some edited cells (Figure 1, panel B).
  • spontaneously generated DSBs within the genome that occur concurrently with CRISPR-Cas9-induced DSBs may also form general chromosomal translocations together with target DSBs (Frock et al., 2015). These general chromosomal translocations can also be captured by PEM-seq, albeit at lower levels and likely not reproducible across different batches of CRISPR-Cas9 treatments.
  • General chromosomal translocations were widely distributed throughout the genome, ranging from 0.51% to 1.96% of total editing results with different baits at 3 days after transfection (Panel E of Figure 1 and Panel D of Figure 9).
  • Table 6 T cell data (corresponding to raw data of Figure 1)
  • This example uses bait primers at the TRAC gene to generate a PEM-seq library.
  • this embodiment detected cutting efficiencies close to those of wild Cas9 ( Figure 9, panel F).
  • this example only detects in the wild-type Cas9 library 3 days after transfection (Fig. 9, panel G), and this result indicates that the editing specificity of these Cas9 variants is higher.
  • similar levels of chromosomal translocations were observed in the PEM-seq libraries of wild-type Cas9 and Cas9 variants ( Figure 1, panel G).
  • the frequency of chromosomal translocations is determined by the interaction strength and frequency of the two DSBs involved (Alt et al., 2013). Since the strength of the interaction between two given DSBs is relatively fixed, this example turns attention to the frequency of CRISPR-Cas9-induced DSBs in chromosomal translocation formation.
  • the repair results after CRISPR-Cas9 cleavage include complete repair, indel and chromosomal translocation, in which the product of complete repair is similar to the uncut target site, which can be repeatedly cut by CRISPR-Cas9 to increase the frequency of DSB ( Figure 2A picture).
  • this example examines the distribution of chromosomal translocation junctions between TRAC and its off-target sites to validate recurrent cleavage by CRISPR-Cas9 .
  • TRAC off-target sites with four mutations within the sgRNA body can generate two types of chromosomal translocation products: non-targetable products, which lose the protospacer-adjacent motif (PAM); retargetable products, whose There is only one mutation at the N of the NGG PAM (panel B of Figure 2).
  • PAM protospacer-adjacent motif
  • retargetable products whose There is only one mutation at the N of the NGG PAM (panel B of Figure 2).
  • chromosomal translocation junctions identified by PEM-seq showed an off-target bias, i.e., only a quarter of the remaining retargetable products were non-targetable (Fig. 2, Panel D).
  • this example also found that the TRAC site also exhibited a similar off-target bias at the off-target site in HEK293T cells ( Figure 2, panel E). And a new TRAC off-target site has an off-target bias of 2 (Panel E of FIG. 2 ).
  • TRBC and PDCD1 also exhibited off-target bias ranging from 2.3 to 3.0 (panel E of Figure 2).
  • This example reanalyzes previous data (Yin et al., 2019) in HEK293T cells using PEM-seq.
  • Off-target bias was widely observed at DNMT1, C-MYC, two loci in the RAG1 gene (RAG1A and RAG1B) in HEK293T cells and at the RAG1A locus in HCT116, U2OS and K562 cells (Fig. A and Table 7).
  • the highest level of off-target bias reached approximately 5.6-fold ( Figure 2, Panel F and Table 7).
  • this example performed CRISPR-Cas9 editing and PEM-seq analysis in G1-arrested cells in which processing of broken ends is restricted to promote the formation of full repairs A product of (Symington and Gautier, 2011). To this end, this example arrests K562 cells in the G1 phase for 36 hours with 5 ⁇ M CDK inhibitor Palbociclib before transfecting CRISPR-Cas9 targeting two sites within the HBA1 or C-MYC gene ( C-MYC1 and C-MYC2) ( Figure 10, panel C).
  • chromosomal translocations With regard to chromosomal translocations, the levels of chromosomal translocations between HBA1 and the three off-target genes were 6.0, 64.0 and 23-fold higher in G1-arrested cells than in circulating cells (Fig. 2G and Fig. 10, panel E). Similar results were obtained at both C-MYC loci in Gl-arrested cells ( Figure 10, panels E and F).
  • Example 5 Cas9-TREX2 inhibits repeat cleavage and chromosomal translocation
  • this example fuses Cas9 with an exonuclease to inhibit repeat cleavage by promoting end processing.
  • this example combined Cas9 at the C-terminus with human 3'-5' exonuclease TREX2 (Cas9X2, Cas9-(G)4S-TREX2 from N-terminus to C-terminus) or nucleic acid-free DNA with H188A mutation Enzymatically active (nuclease-dead TREX2) TREX2 (Cas9X2d, Cas9-(G)4S-nuclease-dead TREX2 from N-terminus to C-terminus) was fused together (Figure 3, panel A; Perrino et al., 2005).
  • T2A-TREX2 Separated forms of Cas9 and TREX2 (T2A-TREX2, Cas9- T2A-TREX2). Plasmids containing these CRISPR genes and sgRNAs were transfected into HEK293T cells for gene targeting, and genomic DNA was harvested 72 hours later to generate a PEM-seq library.
  • Cas9X2d and T2A-TREX2 reduced off-target bias at three HBA1 off-target sites, and Cas9X2 almost eliminated off-target bias at all three sites (panels C and Panel A in 11).
  • all three Cas9-derived enzymes reduced off-target sites and general chromosomal translocation levels for which Cas9X2 ⁇ T2A-TREX2 ⁇ Cas9X2d ⁇ wild-type Cas9 (D in Figure 3 and Figure E).
  • the level of chromosomal translocation at the second off-target site of HBA1 was even reduced to below 0.004% (D in FIG. 3 ).
  • TREX2 Due to its involvement in DNA repair, TREX2 is expressed in many cell types, and ectopic expression of TREX2 has no effect on cell survival or cell cycle (Certo et al., 2012; Mazur and Perrino, 2001).
  • this example generated a Cas9X2 variant with R163A, R165A and R167A mutations (TREX2-3R) to eliminate the DNA binding activity of TREX2 (Perrino et al., 2008). Then, this example purified TREX2, TREX2-3R and its fusion form with Cas9 to carry out the in vitro digestion assay of 38-nt oligonucleotides (panel A in FIG. 12 ).
  • TREX2-3R showed a substantial reduction in exonuclease activity towards oligonucleotides ( Figure 12, panel B). Digestion products shorter than 37nt were detected for 0.5nM Cas9X2 at 21 minutes, but no digestion products were detected for 2.7nM Cas9-TREX2-3R (Cas9TX) at 63 minutes (Panel A of Figure 4), which means that Cas9TX Potentially better security.
  • Cas9TX was applied to genome editing in HEK293T cells, and PEM-seq analysis was performed. Despite losing the DNA-binding ability of TREX2, Cas9TX could still precisely localize to HBA1 and C-MYC target sites, and showed slightly higher editing efficiency than Cas9 at these two sites, as did Cas9X2 (Fig. Figure B of 4). Both Cas9X2 and Cas9TX effectively eliminated off-target bias and reduced total chromosomal translocations, but Cas9TX performed better than Cas9X2 (panel C in Figure 4, panels C and D in Figure 12). This example then tested the other 12 sites in HEK293T cells.
  • Example 7 The level of chromosomal translocation induced by Cas9TX is comparable to that induced by base editors
  • Cytosine base editor (CBE) and adenine base editor (ABE) systems are also currently being developed to induce point mutations at target sites to complete gene editing (Gaudelli et al., 2017; Komor et al., 2016). Since CBE or ABE do not generate DSBs, chromosomal translocations are rarely formed in base editing systems.
  • this example uses CRISPR-Cas9, CRISPR-Cas9TX, cytosine base editor BE4max and adenine base editor ABEmax (Koblan et al., 2018 ), targeting five target sites within EMX1, DNMT1, C-MYC, RAG1, and BCL11A genes in HEK293T cells, and then using PEM-seq for chromosomal translocation detection.
  • BE4max can efficiently target multiple cytosines, while ABEmax can target multiple adenines within the editing window, as shown in the RAG1C library in panel A of Figure 5.
  • ABEmax could not target the EMX1 and C-MYC2 sites due to the lack of adenine in the editing window.
  • Cas9 induced massive indels, with the highest base loss near the break site.
  • Cas9TX showed accumulation of base loss at the break end containing the 17bp truncated sgRNA ( Figure 5, panel A), which was due to end processing of resident Cas9TX after cleavage (Brinkman et al., 2018 ).
  • Cas9 induced a large number of chromosomal translocations in all five sites, while Cas9TX showed very low levels of chromosomal translocations (panels C and D in Figure 5 and panel B in Figure 13), which is consistent with the above results.
  • both BE4max and ABEmax induced very low levels of general chromosomal translocations at the tested sites, but above background levels (panels B and C in Figure 5 and panel B in Figure 13).
  • the level of chromosomal translocation in Cas9TX was lower than 0.45% at all tested sites, it was still slightly higher than that of the two base editors (Panel C in Figure 5 and Panel C in Figure 13).
  • this example expresses Cas9-P2A-mCherry or Cas9TX-P2A-mCherry without sgRNA in HEK293T cells by plasmid transfection.
  • P2A is a self-cleaving peptide that ensures that cells expressing Cas9 or Cas9TX are labeled with mCherry.
  • this example marked the DSB signal in the cells by anti-H2AX antibody.
  • the topoisomerase inhibitor etoposide induced a large number of DSBs labeled with ⁇ H2AX ( Figure 6, panels A and B; Canela et al., 2017).
  • the levels of DSBs were not significantly elevated in cells expressing Cas9 and Cas9TX ( Figure 6, panels A and B), indicating that both Cas9 and Cas9TX displayed undetectable nonspecific genome-wide damage activity in the absence of sgRNA .
  • this example tested the effect of Cas9TX on other DSBs by co-expressing AsCas12a and Cas9TX with crRNA for AsCas12a, targeting the C-MYC gene (C-MYC3) in HEK293T cells.
  • PEM-seq analysis was performed with bait primers at the AsCas12a target site, and it was found that the editing efficiency of AsCas12a was relatively higher when co-expressed with Cas9TX than with Cas9 (18.8% vs. 21.5%; Figure 6, panel C) .
  • AsCas12a:C-MYC3 had fewer chromosomal translocations when co-expressed with Cas9TX than with Cas9 (1.5% vs. Chromosomal translocation expressing editing enzymes.
  • this example also used PEM-seq with bait primers at the identified Cas9:C-MYC2 off-target sites to examine the effect of Cas9TX on cleavage at the off-target sites.
  • this embodiment detected a higher editing frequency at the off-target site of Cas9TX, however, the two are close and proportional to the editing efficiency of the MYC2 target site ( Figure 6, panel D).
  • the MYC2 off-target of Cas9TX showed 14.6-fold fewer chromosomal translocations compared to Cas9 (panel E of FIG. 6 ).
  • This example also used tracking of indels by decomposition (TIDE) to roughly measure the editing frequency of HEK293T cells targeted by VEGFA or EMX1 at multiple off-target sites, most of which showed an increase in the editing frequency of Cas9TX, but It is also directly proportional to the editing efficiency of the target site (Fig. 6, panel F).
  • this example first transduced retrovirus encoding CD19-41BB-CAR (Kawalekar et al., 2016) into activated T cells, and then passed RNP
  • the TRAC, TRBC or PDCD1 gene was edited using Cas9 or Cas9TX (Panel A of Figure 7).
  • the transduction efficiencies of the CAR retroviruses were very close (Figure 7, panel B).
  • CRISPR-Cas9-induced DSBs undergo different DNA repair pathways to generate a range of DNA repair outcomes.
  • the non-homologous end joining (NHEJ) pathway directly joins together two intact or lightly processed break ends to generate either full rejoined products or minor insertions/deletions (Betermier et al., 2014). It is estimated that more than 50% of repairs after CRISPR-Cas9 cleavage result from complete recombination of sequencing data from two adjacent target sites (Brinkman et al., 2018; Guo et al., 2018). Complete rejoining is indistinguishable from the target sequence and can be repeatedly cleaved by CRISPR-Cas9.
  • chromosomal translocations can occur not only between Cas9-induced DSBs but also between general DSBs generated during various cellular events (Alt et al., 2013; Casellas et al., 2016 ; Nussenzweig and Nussenzweig, 2010; Tubbs and Nussenzweig, 2017).
  • Chromosomal translocations have been widely observed in many types of CRISPR-Cas9-edited cells (Eyquem et al., 2017; Liu et al., 2017; Mandal et al., 2014; Ren et al., 2017; Stadtmauer et al ., 2020). Chromosomal translocations are estimated to occur between the two target genes in CRISPR-Cas9-targeted T cells at a frequency of one in 50-300 edited T cells roughly calculated from PEM-seq (Fig. 1C).
  • chromosomal translocations involving strong enhancers from the antigen receptor locus or c-Myc have been extensively studied to be able to drive tumorigenesis in developing lymphocytes (Alt et al., 2013; Lieber, 2016; Nussenzweig and Nussenzweig, 2010).
  • chromosomal translocations generated during CRISPR-Cas9 targeting of CCR5 or B2M genes in hematopoietic stem and progenitor cells ( Liu et al., 2017; Mandal et al., 2014; Ren et al., 2017; Stadtmauer et al., 2020) also pose a threat to the genomic integrity of stem cells and affect the circulatory and immune systems.
  • TREX2 and Cas9 can also increase cutting efficiency, as previously reported (Cermak et al., 2017), but cannot suppress chromosomal translocations as efficiently as Cas9TX in most editing situations, which may be due to low Efficiency arises from timely end-processing, which may result in leakage of complete repair products.
  • the split form of Cas9 and TREX2 is not optimized for RNP delivery, whereas, as shown here, Cas9TX is compatible with RNP and the small size (236 amino acids) of TREX2-3R can also package Cas9TX into a split gland.
  • Related viruses AAV; Chew et al., 2016).
  • fused TREX2 should be able to increase editing efficiency and inhibit chromosomal translocation of other blunt-ended or 3′-end gene editing enzymes, since TREX2 prefers these types of broken ends.
  • CRISPR-Cas9TX nearly eliminated chromosomal translocations between TRAC, TRBC, and PDCD1 target sites, and had no detectable effect on CAR lethality, outperforming CRISPR-Cas9 (Fig. 7).
  • high-fidelity Cas9 variants were unable to suppress translocations despite their higher editing specificity.
  • BE4max and ABEmax are still able to induce low levels of chromosomal translocations, as gaps can be converted to DSBs at low frequency (Tubbs and Nussenzweig, 2017).
  • CRISPR-Cas9TX induced only higher levels of general chromosomal translocations than BE4max or ABEmax (Fig. 5C).
  • base editors can also target RNA (Grunewald et al., 2019; Zhou et al., 2019), whereas CRISPR-Cas9TX does not have this ability.
  • CRISPR-Cas9TX ensures relatively good genome integrity like base editors.
  • the inventors successfully applied the fusion protein of the present invention to transforming CAR-T tumor therapy. Knocking out the coding genes of TCR and PD-1 in the process of CAR-T by using the fusion protein of the present invention can effectively improve the targeting and survival cycle of CAR-T.
  • Cas9 there are about 300 chromosomal translocations in 100,000 edited cells in the CAR-T to be infused back.
  • the level of chromosomal translocations had dropped to baseline levels, that is, they had basically disappeared.
  • Cas9TX can significantly improve the gene safety in the process of single-gene and multi-gene editing, and has great application prospects in the clinical application of gene editing.
  • TREX2 mutant TREX2-3R amino acid sequence (SEQ ID NO.1):
  • TREX2 mutant nucleotide sequence (SEQ ID NO.4):

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Abstract

本发明涉及融合蛋白以及其使用方法。融合蛋白包含第一蛋白和第二蛋白，其中第一蛋白是Cas,如Cas9、Cas12a、Cas12b、Cas12e以及TALEN或ZFN，第二蛋白是核酸外切酶或者核酸内切酶，如TREX2、TREX1、APE1、Artemis、CtIP、Exo1、Mre11、RAD1、RAD9、Tp53、WRN、外切核酸酶V、T5外切核酸酶或T7外切核酸酶。第一蛋白与第二蛋白是直接连接的或者通过接头连接。本发明的融合蛋白可以保证基因编辑过程中的基因组稳定性，降低单基因或多基因编辑过程中的染色体结构变异的产生。

Description

融合蛋白以及其使用方法
本发明要求申请号为202110900953.3，申请日为2021年08月06日，专利名称为“融合蛋白以及使用方法”的中国专利申请的全部优先权。
技术领域
本发明涉及蛋白质领域，具体地涉及用于基因编辑的融合蛋白。
背景技术
基因编辑安全性一直是影响基因编辑在临床上应用的最大障碍。脱靶活性是比较受关注的一个影响基因编辑安全性的方面，而且多种高保真酶的开发也能降低Cas9等酶的脱靶活性(Kleinstiver et al.,2016；Schmid-Burgk et al.,2020；Slaymaker et al.,2016；Yin et al.,2019)。DNA修复产生的染色质结构变异也是Cas9等基因编辑工具酶进行编辑过程中必然产物，常常与癌症发生偶联在一起。但是由于缺乏有效的鉴定工具，目前对其认识主要是对偶然发现的产物的报道。数篇研究报道了基因编辑产物中包含染色体大片段缺失和染色体易位，这成为阻碍基因编辑临床应用的一个重要方面(Cullot et al.,2019；Egli et al.,2018；Kosicki et al.,2018；Shin et al.,2017)。
针对脱靶效应，目前已经有了通过利用Cas9高保真突变体来减弱的成熟方案，但对于染色体异常结构的产生，从该领域发展至今尚缺乏很好的应对策略。染色体异常结构是由细胞内的DNA修复通路决定，常常与癌症的发生偶联在一起，是一种非常危险的副产物。目前国际上缺乏可以定量检测这些结构的方法。发明人先前的PEM-seq(Primer-extension-mediated sequencing)(Yin et al.,2019)方法可以检测各种DNA修复异常结构。
此外，目前医学以及科研工作者经常将T淋巴细胞进行分离，活化，离体扩增，并注入患者体内以进行过继T细胞免疫疗法(Rosenberg et al.,2008；Laskowski and Rezvani,2020)。除了使用患者自身或其他健康供体的原始T细胞外，T细胞最终还可以经过工程化改造以容纳合成的嵌合抗原受体(Chimeric antigen receptor,CAR)或新抗原特异性T细胞受体(T cell receptor,TCR)以增强其抗肿瘤功能(Morgan et al.,2006；Kochenderfer and Rosenberg,2013；Porter et al.,2011；June and Sadelain,2018)。CAR T细胞通过使用抗体的抗原结合结构域直接识别肿瘤细胞进行攻击，从而绕过了主要组织相容性复合物(Major histocompatibility complex,MHC)呈递肿瘤抗原的要求(Kalos and June,2013；Rafiq et al.,2020)。CAR和TCR T细胞都已显示出在癌症治疗中的巨大潜力，特别是对于B细胞恶性肿瘤，除数种市售CAR T疗法外，数百项临床试验已获批准(Wei et al.,2020；Manfredi et al.,2020)。
为了进一步提高工程化改造的CAR或TCR T细胞的特异性和持久性，相关工作利用基因编辑失活导致T细胞功能障碍或衰竭的基因，以开发下一代过继T细胞免疫疗法(Hendriks et al.,2020)。与之相关，相关工作已靶向恒定区中的外显子处靶向TCRα和β链基因TRAC和TRBC，以减少TCR T细胞中转导的肿瘤特异性TCR的错配并增强CAR T细胞中的肿瘤排斥(Stadtmauer et al.,2020；Eyquem et al.,2017)，以及免疫检查点因子编程化细胞死亡蛋白1(PD-1)的缺失使工程化的CAR或TCR T细胞在输注后能够在患者体内持续存在(Stadtmauer et al.,2020；Ren et al.,2017a)。消除TRAC，TRBC或人白细胞抗原I类(HLA-1)亚基基因β-2微球蛋白(B2M)可以在植入同种异体T细胞时降低移植物抗宿主疾病(GVHD)的风险(Liu et al.,2016；Ren et al.,2017)。此外，其他基因的破坏也能够增强工程化的T细胞，并有数十项临床试验正在进行(Fraietta et al.,2018；Hendriks et al.,2020)。
虽然锌指核酸酶(Zinc finger nucleases,ZFN)，转录激活因子样效应子核酸酶(Transcription activator-like effector nucleases,TALEN)和成簇规则间隔短回文重复序列(CRISPR)-Cpf1已成功应用于破坏CAR T细胞中的单个或多个基因(Torikai et al.,2012；Poirot et al.,2015；Qasim et al.,2017；Dai et al.,2019)，但是由于CRISPR-Cas9的多重基因组编辑的高度可行性，目前大多数过继T免疫疗法临床试验都已倾向使用CRISPR-Cas9(Hendriks et al.,2020)。CRISPR-Cas9在被靶向的基因处产生DNA双链断裂(DNA double-stranded breaks,DSB)，以高效地同时失活工程化T细胞中的多个基因(Stadtmauer et  al.,2020；Liu et al.,2016；Ren et al.,2017)。然而，在T细胞中进行多重基因组编辑时，两个不同靶位点之间通常会形成染色体易位(Stadtmauer et al.,2020；Liu et al.,2016；Ren et al.,2017)。据估计，对于诱导的DSB或来自细胞内源生理活动的DSB，染色体易位发生的频率为千分之一至百分之一(Roukos et al.,2013；Hu et al.,2016；Tubes and Nussenzweig,2017)。进一步而言，人们在T细胞急性成淋巴细胞白血病中经常观察到染色体易位，并且超过三分之一以上涉及TCR基因座(Graux et al.,2006；Cauwelier et al.,2006)。基因编辑过程中产生的染色体易位阻碍了其进一步的应用。
因此，本领域中迫切需要解决基因编辑安全性的基因编辑工具酶。
发明内容
发明人在前期研究中发现，在制造用于过继细胞疗法的工程化T细胞期间，在多重靶向位点及其脱靶位点中形成了大量的染色体易位。而这是由于CRISPR-CAS系统切割的完美修复产物能够被重复切割，进一步增加了切割频率而导致的。上述染色体易位的存在也进一步对基因编辑技术的安全性提出了巨大的挑战。
在此基础上，发明人创造性的发现，如果将Cas蛋白与核酸酶(例如核酸外切酶)融合形成Cas内外双切酶，就可以极大的降低完美修复的出现频率，从而大大降低了CRISPR-CAS系统导致的染色体易位的水平。也就是说，将具有外切核酸酶活性的蛋白质与RNA引导的内切核酸酶如Cas蛋白连接(直接连接或通过接头连接)得到的融合蛋白可以减少或消除细胞修饰(例如利用CRISPR-Cas9的细胞修饰)中的染色体易位或染色体缺失。例如，将CAS9与TREX2核酸外切酶融合，能够将染色体易位水平降低几十倍，达到仅仅略高于碱基编辑器或与之相当的水平。在CRISPR技术中利用本发明的融合蛋白进行嵌合抗原受体(CAR)T细胞中的多重基因编辑，并且在输注之前几乎消除了有害的涉及TCR的染色体易位。此外，在CRISPR技术中使用本发明的融合蛋白还抑制染色体易位并提高单基因编辑的编辑效率。因此，本发明的融合蛋白是一种有效的基因组编辑工具，具有更高的安全性，并可同时维持或提升基因编辑效率。
在一方面，本发明提供了该融合蛋白包含第一蛋白和第二蛋白，其中
其中第一蛋白是Cas蛋白、TALEN或ZFN；
第二蛋白是核酸外切酶或核酸内切酶，例如TREX2(three prime repair exonuclease 2)、TREX1(three prime repair exonuclease 1)、APE1(apurinic/apyrimidinic endodeoxyribonuclease 1)、Artemis蛋白、CtIP(CtBP-interacting protein)、Exo1(exonuclease 1)、Mre11(MRE11 homolog,double strand break repair nuclease)、RAD1(RAD1 checkpoint DNA exonuclease)、RAD9蛋白、Tp53(tumor protein p53)、WRN蛋白、外切核酸酶V、T5外切核酸酶或T7外切核酸酶。
在一个实施方案中，Cas蛋白是Cas9、Cas12a、Cas12b或Cas12e蛋白。
在一个实施方案中，第一蛋白与第二蛋白是直接连接的或者通过接头连接。第一蛋白可以在融合蛋白的C端或N端。
在一个实施方案中，Cas9为SpCas9或SaCas9。在一个实施方案中，Cas12a是AsCas12a，Cas12e为PlmCas12e。
在一个实施方案中，接头是(G)n、(GGGGS)n、(EAAAK)n、(XP)n或XTEN接头，其中n是大于等于1的整数，X是任何氨基酸，优选丙氨酸、赖氨酸或谷氨酸。在一个实施方案中，接头是(G)4S接头。在一个实施方案中，n是2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90或100，或其间的任何整数。在一个实施方案中，X是任何天然存在的氨基酸或非天然存在的氨基酸。
在一个实施方案中，TREX2蛋白包含SEQ ID NO.8的氨基酸序列或其中引入R163A、R165A和R167A中一个或多个的氨基酸序列，或者与它们具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。在一个实施方案中，TREX2蛋白包含：
(1)SEQ ID NO.1的氨基酸序列；
(2)SEQ ID NO.1的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或
(3)与SEQ ID NO.1的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一个实施方案中，Cas9蛋白包含：
(1)SEQ ID NO.2的氨基酸序列；
(2)SEQ ID NO.2的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或
(3)与SEQ ID NO.2的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一个实施方案中，融合蛋白包含
(1)SEQ ID NO.3的氨基酸序列；
(2)SEQ ID NO.3的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或
(3)与SEQ ID NO.3的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
Cas12e蛋白包含：
(1)SEQ ID NO.89的氨基酸序列；
(2)SEQ ID NO.89的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或
(3)与SEQ ID NO.89的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
Cas12a蛋白包含：
(1)SEQ ID NO.91的氨基酸序列；
(2)SEQ ID NO.91的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或
(3)与SEQ ID NO.91的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在另一个方面，本发明提供了核酸，该核酸编码根据本发明的融合蛋白。
在另一个方面，本发明提供了载体，该载体包含根据本发明的核酸。
在另一个方面，本发明提供了宿主细胞，该宿主细胞包含本发明的载体。
在另一个方面，本发明提供了制备本发明的融合蛋白的方法，其包括以下步骤：
(1)在适合于培养宿主细胞的条件下培养本发明的宿主细胞；以及
(2)收获本发明的融合蛋白。
在另一个方面，本发明提供了组合物，该组合物包含本发明的融合蛋白和sgRNA。在一个实施方案中，组合物是核糖核蛋白复合物。在一个实施方案中，核糖核蛋白复合物是CRISPR-Cas系统。在一个实施方案中，CRISPR-Cas系统是CRISPR-Cas9、CRISPR-Cas12a、CRISPR-Cas12b或者CRISPR-Cas12e系统。
在另一个方面，本发明提供了遗传修饰细胞的方法，所述方法包括将根据本发明的融合蛋白、核酸、载体或组合物导入细胞的步骤。优选地，细胞是T细胞或HSC细胞。优选地，细胞是TCR T细胞、NK细胞或CAR-T细胞。
在一个实施方案中，本发明提供了一种减少或者消除利用CRSPR-Cas进行基因编辑时产生的染 色体结构变异的方法，其包括将本发明的融合蛋白、核酸、载体或者组合物导入目标细胞的步骤。优选地，细胞是T细胞或HSC细胞，优选地，其中所述细胞是TCR-T细胞或CAR-T细胞。
在一个实施方案中，本发明提供了一种基因编辑方法，其包括将本发明的融合蛋白、核酸、载体或者组合物导入目标细胞的步骤，优选地，其中所述细胞是T细胞或HSC细胞。优选地，细胞是TCR-T细胞或CAR-T细胞。
在一个实施方案中，组合物能够减少或消除基因编辑过程中的染色体异常结构。
在一个实施方案中，基因选自Oct4、MYH7、TRAC、PDCD1、TRBC、UROS、Fzd3、TLX1(HOX11)、TLX3(HOX11L2)、HOXA簇、HAL1、HAL2、LYL1、BHLHB1、LMO1、LMO2、LCK、NOTCH1、CCND2、SIL/TAL1、CALM/AF10、MLL/ENL、MLL/AF6、MLL/AF10、MLL/AFX1、MLL/AF4、NUP214/ABL1、EML1/ABL1、ETV6(TEL)/ABL1、ETV6(TEL)/JAK2、BCR/ABL1、NUP98/RAP1GDS1、B2M、CCR5或VEGFA。
在一个实施方案中，染色体异常结构是染色体易位和/或染色体缺失。
在一个实施方案中，染色体缺失是染色体的大片段缺失。在一个实施方案中，染色体的大片段缺失是100bp-4Mb染色体片段缺失。在一个实施方案中，染色体的大片段缺失可以是200bp、300bp、400bp、500bp、600bp、700bp、800bp、900bp、1kb、5kb、10kb、20kb、30kb、40kb、50kb、100kb、200kb、300kb、500kb、600kb、700kb、800kb、1Mb、2Mb或3Mb的染色体的大片段缺失。
在一个实施方案中，基因编辑通过CRISPR-Cas系统进行，优选通过CRISPR-Cas9系统、CRISPR-Cas12a、CRISPR-Cas12b或者CRISPR-Cas12e进行。
在另一个方面，本发明提供了细胞，其已经通过本发明的方法进行了修饰。在一个实施方案中，所述细胞是T细胞或HSC细胞。在一个实施方案中，所述细胞是TCR T细胞或CAR-T细胞。
在另一个方面，本发明提供了使用本发明的细胞治疗疾病的方法。在另一个方面，本发明提供了本发明的细胞在制备药物中的用途，所述药物用于治疗需要该细胞的受试者中的疾病。在一个实施方案中，疾病是遗传疾病，例如癌症、自身免疫疾病或眼病。在一个实施方案中，杜氏肌营养不良症、β-地中海贫血、镰状细胞贫血、膀胱癌、髓母细胞瘤、子宫颈癌、乙型肝炎、EBV感染、HIV感染和/或隐孢子虫感染、脉络膜血管增生或黑曚症。在一个实施方案中，使用本发明的细胞可以用于细胞疗法。在一个实施方案中，细胞是自体的或者同种异体的。
在另一个方面，本发明提供了本发明的融合蛋白、核酸、载体或组合物减少或消除基因编辑过程中的染色体异常结构的用途。优选地，其中基因选自Oct4、MYH7、TRAC、PDCD1、TRBC、UROS、Fzd3、TLX1(HOX11)、TLX3(HOX11L2)、HOXA簇、HAL1、HAL2、LYL1、BHLHB1、LMO1、LMO2、LCK、NOTCH1、CCND2、SIL/TAL1、CALM/AF10、MLL/ENL、MLL/AF6、MLL/AF10、MLL/AFX1、MLL/AF4、NUP214/ABL1、EML1/ABL1、ETV6(TEL)/ABL1、ETV6(TEL)/JAK2、BCR/ABL1、NUP98/RAP1GDS1、B2M、CCR5或VEGFA。优选地，染色体异常结构是染色体易位和/或染色体缺失。
优选地，染色体缺失是100bp-4Mb的染色体片段缺失。
优选地，所述基因编辑通过CRISPR-Cas系统进行，优选通过CRISPR-Cas9系统、CRISPR-Cas12a、CRISPR-Cas12b或者CRISPR-Cas12e进行。
在本文中，基因编辑可以是单基因编辑或多重基因编辑。
在另一个方面，本发明提供了融合蛋白、核酸、载体或组合物在保证基因编辑过程中的基因组稳定性、提高CAR-T构建过程中T细胞的基因组稳定性或防止改造的CAR-T细胞发生瘤变中的用途。该用途通过降低染色体异常结构实现。
本发明的优点包括：
1.与单独的核酸酶，例如TALEN或ZFN，或Cas蛋白，例如现有的Cas9相比，本发明的融合 蛋白可以显著减弱或消除基因编辑过程中的染色体大片段缺失；
2.与单独的核酸酶，例如TALEN或ZFN，或Cas蛋白，例如现有的Cas9相比，本发明的融合蛋白可以显著减弱或消除基因编辑过程中的染色体易位；
3.与单独的核酸酶，例如TALEN或ZFN，或Cas蛋白，例如现有的Cas9相比，本发明的融合蛋白在实现上述1和2点的同时，具有相当甚至略高的基因编辑效率；
4.与现有CAR-T技术相比，可以在保持操作不变、编辑效率不变的情况下，降低所改造的CAR-T细胞的瘤变的可能性，具有显著更高的安全性。
附图说明
图1：Cas9在人T细胞中介导多重基因编辑过程中的染色体易位。
A.在人T细胞中，在Cas9 RNP进行多重基因编辑期间评估染色体易位的示意图。B.PEM-seq检测转染后第3、7和14天，人T细胞中TRAC，TRBC和PDCD1三个位点的编辑效率。该数据来自三个重复的平均值±SD，分别由“圆形”，“三角形”和“正方形”表示。数据检验利用两尾t检验，*p<0.05和**p<0.01。C.Circos图表明了PEM-seq检测到的转染后3天，人T细胞中TRAC，TRBC，PDCD1和TRAC脱靶位点之间的染色体易位。顺时针方向指示染色体排列方向，着丝粒至端粒。染色体易位的连线上面标注了三个重复的平均值，连线的箭头方向指示诱饵与猎物的方向。D.Cas9 RNP转染3、7和14天后，PEM-seq在人T细胞中检测到的TRAC，TRBC和PDCD1之间的染色体易位百分比。染色体易位被表示为诱饵-猎物，例如：TRAC-TRBC代表TRAC与用位于TRAC的诱饵引物克隆的TRBC之间的染色体易位。来自三个重复的平均值±SD。E.PEM-seq检测人T细胞中转染后3、7和14天从所示基因座克隆的一般染色体易位的百分比。来自三个重复的平均值±SD。在每个数据点上方标出平均值。F.针对Cas9 RNP转染后3天，从人T细胞中TRAC，TRBC和PDCD1鉴定的总染色体易位，使用Enrichr的KEGG进行基因注释(maayanlab.cloud/Enrichr/)。横轴表示所示途径中的基因数目。G.在转染后3天，PEM-seq检测从TRAC克隆的人T细胞中Cas9和相应的变体酶诱导的染色体易位的百分比。
图2：CRISPR-Cas9重复切割增强脱靶偏倚。
A.CRISPR-Cas9编辑结果和重复切割的示意图。Cas9通常可以产生完美重新连接，插入缺失和染色体易位。完美重新连接或一些轻度突变的修复产物可以被CRISPR-Cas9重复切割。B.TRAC中靶位点与脱靶位点之间的潜在染色体易位产物的DNA序列。箭头指示用于生成PEM-seq文库的诱饵引物。根据所示的诱饵引物，可形成两种类型的染色体易位产物“可再靶向”和“不可靶向”。浅灰色字母表示与TRAC中靶位点序列不匹配的碱基。C.Cas9：TRAC对TRAC的中靶位点，脱靶位点和染色体易位产物(如图2的B图所示)的体外消化。黑色箭头表示400bp大小的DNA标志物。被切割的DNA由黑色括号指示。D.PEM-seq检测的TRAC中靶位点克隆的人T细胞中“可再靶向”和“不可靶向”染色体易位产物的百分比。接合数目在括号中。脱靶位点偏倚由“不可靶向”与“可再靶向”的比值计算。E.PEM-seq检测到的HEK293T细胞中TRAC，TRBC和PDCD1脱靶的脱靶偏倚。虚线表示比率＝1。F.PEM-seq检测HEK293T细胞中RAG1A，RAG1B，DNMT1-1和C-MYC1基因座的脱靶位点处的脱靶偏倚。数据来自三个重复的平均值±SD。虚线表示比率＝1。x轴上的数字表示从强到弱的脱靶顺序。G.K562细胞中HBA1基因座的三个脱靶位点(OT1至OT3)的脱靶染色体易位(上)和脱靶偏倚(下)的百分比。横条上标出倍数变化和脱靶偏倚。具有很少接合的脱靶位点以*突出显示。
图3：通过将TREX2与Cas9融合来减少重复切割和染色体易位。
A.Cas9-TREX2融合蛋白的示意图。Cas9和TREX2通过GGGGS接头连接。R163，R165和R167对于DNA结合活性至关重要，而H188对于TREX2的核酸外切酶活性是必需的。B.PEM-seq检测HEK293T细胞中HBA1基因座处的Cas9，Cas9-T2A-TREX2，Cas9X2和Cas9X2d的编辑效率。灰色条表示缺失的比率，深色条表示插入的比率。在每个条上标记缺失率和总编辑效率。C和D.PEM-seq 检测到的HEK293T细胞中HBA1处Cas9，Cas9-T2A-TREX2，Cas9X2和Cas9X2d的脱靶偏倚(C)和脱靶染色体易位比(D)。脱靶偏倚和脱靶染色体易位的倍数变化位于每个条形的顶部。E.用于HEK293T细胞中HBA1处Cas9，Cas9-T2A-TREX2，Cas9X2和Cas9X2d PEM-seq文库的染色体易位的Circos图。浅色箭头指示靶向位点，黑色箭头指示已识别的脱靶位点。脱靶染色体易位的百分比用浅色标记，并且一般染色体易位的百分比用黑色标记。F-I.对于HEK293T细胞中的Cas9，Cas9-T2A-TREX2，Cas9X2和Cas9X2d PEM-seq文库，编辑效率(F)，编辑事件中的缺失率(G)，脱靶染色体易位的百分比(H)，一般染色体易位的百分比(I)。n＝10，使用的基因座是DNMT1-1，DNMT1-2，EMX1，HBA1，C-MYC1，C-MYC2，CMYC3，RAG1A，RAG1B和RAG1C。注意，对于Cas9X2而言，在几个基因座中未检测到脱靶染色体易位。Wilcoxon检验，*p<0.05，**p<0.01，n.s表示无显著性。
图4：Cas9TX的表现超出Cas9X2的表现。
(A)体外切割测定法测定，在不使用sgRNA的情况下，Cas9X2和Cas9TX对38-mer寡核苷酸的消化。将指定量的蛋白质与7.5nM 38-nt寡核苷酸温育指定时间。NC，BSA作为阴性对照。B和C.在HEK293T细胞中PEM-seq检测HBA1和C-MYC2处的Cas9，Cas9X2，Cas9TX的编辑效率(B)，脱靶染色体易位百分比(C，上部)和脱靶偏倚(C，下部)。如图12的C图所示，所示的HBA1脱靶位点是OT3。脱靶染色体易位的倍数变化和计算出的脱靶偏倚位于每个条形的顶部。D-F.PEM-seq检测到的HEK293T细胞中Cas9和Cas9TX的编辑效率(D)，脱靶染色体易位的百分比(E)和一般染色体易位的百分比(F)。n＝14，使用的基因座是DNMT1-1，DNMT1-2，HBA1，C-MYC1，C-MYC2，C-MYC3，RAG1A，RAG1B，RAG1C，PTEN，TP53，TRAC，TRBC和PDCD1。Wilcoxon检验，**p<0.01和***p<0.001。
图5：Cas9TX将染色体易位抑制到碱基编辑器的水平。A.PEM-seq检测到的RAG1C基因座处，BE4max，ABEmax，Cas9和Cas9TX的断裂位点周围的编辑模式。箭头表示假定的断裂部位。在核苷酸分辨率上显示突变，缺失和插入的累积水平。B和C.对于HEK293T细胞中EMX1，C-MYC2，DNMT1-2，RAG1C和BCL11A处，PEM-seq检测到的Cas9，Cas9TX，BE4max和ABEmax的编辑效率(B)和一般染色体易位百分比(C)。值得注意的是，EMX1和C-MYC2位点不能被ABEmax靶向。D.Circos图显示了HEK293T细胞中RAG1C处Cas9，Cas9TX，BE4max和ABEmax的染色体易位分布。红色箭头指示RAG1C靶位点。Circos图中的数字显示了一般染色体易位的百分比。
图6：评估Cas9TX对基因组稳定性的影响。A.依托泊苷，Cas9或Cas9TX处理的HEK293T细胞的γH2AX的免疫荧光。该图片为共聚焦显微镜在转染后24小时拍摄图像。Cas9和Cas9TX均用P2A-mCherry标签表达。单独的mCherry是阴性对照。B.依托泊苷，Cas9或Cas9TX处理的每个HEK293T细胞中γH2AX焦点的统计数据。两尾t检验，****p<0.00001；n.s，无显著性。C.PEM-seq评估Cas9和Cas9TX对HEK293T细胞中AsCas12a:C-MYC3的编辑效率和染色体易位水平的影响。D.在HEM293T细胞中，PEM-seq检测到的C-MYC2以及鉴定出的C-MYC2脱靶位点处的Cas9和Cas9TX的编辑效率。来自三个重复的平均值±SD。顶部显示了C-MYC2和C-MYC2脱靶的DNA序列。错配的DNA为浅色。E.HEK293T细胞中通过PEM-seq从C-MYC2脱靶位点克隆的染色体易位百分比。来自三个重复的平均值±SD。两尾t检验，**p<0.01。F.在VEGFA和EMX1的靶向和脱靶处的Cas9和Cas9TX的编辑频率。编辑效率由TIDE评估。从Cas9TX到Cas9的倍数变化位于顶部。平均值±SD。
图7:Cas9TX在CAR-T细胞中消除了染色体易位。A.Cas9或Cas9TX RNP制备CAR-T细胞的示意图。从人脐带血获得的人T细胞通过抗CD3/CD28活化3天，由携带抗CD19 scFV的慢病毒感染，然后进行Cas9/Cas9TX RNPs转染。在转染后3天进行CAR-T杀伤测定法，并在转染后3、7和14天通过PEM-seq分析TRAC，TRBC和PDCD1之间的染色体易位。B.FACS检测到的由携带CAR-CFP的慢病毒感染的T细胞的百分比。3次重复的平均值±SD；n.s，无显著性。C.通过PEM-seq 检测到的转染后3天的TRAC，TRBC和PDCD1基因处的编辑效率。3次重复的平均值±SD；n.s，无显著性。D.流式细胞术结果检测转染后3天的TCR表达水平。NC是未进行RNP编辑的阴性对照。E和F.Circos图表示通过PEM-seq检测到的RNP转染后第3天，Cas9(E)或Cas9TX(F)在TRAC，TRBC，PDCD1和TRAC脱靶之间的染色体易位。星号表示已识别的TRAC脱靶位点，如图1C的图例中所述。G-I.转染后3、7和14天根据PEM-seq的测定，从TRAC克隆的TRAC-TRBC(G)，TRAC-PDCD1(H)的由Cas9或Cas9TX诱导的染色体易位和TRAC一般染色体易位(I)的比率。显示了三个重复的平均值。两尾t检验，*p<0.05，**p<0.01。J.通过CD19 +K562杀伤测定法评估Cas9和Cas9TX编辑的CAR-T细胞的杀伤能力。T代表靶细胞，E代表效应细胞。
图8：在CAR-T细胞工程中的多重基因编辑过程中，Cas9TX减少了大的染色体片段缺失。A.基因组编辑过程中大缺失形成的示意图。DNA切除，随后进行Cas9切割，导致DNA丢失，导致大规模DNA缺失。B-D PEM-seq检测，在转染后3天，7天和14天，对于TRAC(A)和PDCD1(B)和TRBC(C)，Cas9和Cas9TX得到的大染色体片段缺失的百分比。数据来自三个重复的t检验，**p<0.01。
图9：A.PEM-seq鉴定CRISPR-Cas9编辑结果的示意图。B.在SDS PAGE上显示Cas9和Cas9高保真度变体的纯化。C.在转染后3、7和14天通过人T细胞的巢式PCR检测TRAC，TRBC和PDCD1之间的染色体易位。在第一轮PCR中使用F1和R1，回收产物用于第二轮PCR，第二轮PCR使用F2和R2进行。具体见方法部分。D.在转染后3、7和14天PEM-seq在人T细胞中检测到的指定染色体易位的平均百分比。E.在转染后3天，在人T细胞中PEM-seq检测，从TRAC，TRBC或PDCD1克隆的全基因组染色体易位模式。星号表示TRAC，TRBC，PDCD1靶向以及鉴定的TRAC脱靶。F.在转染后3天，PEM-seq检测，TRAC基因处的Cas9和指定的高保真度变体的编辑效率。注：此处Cas9文库是图1的B图中所示的三个重复之一。G.在转染后3天，人T细胞中的Cas9和指定高保真度变体在已鉴定的TRAC脱靶处的染色体易位接合的分布。
图10：A.PEM-seq检测到的HCT116，K562和U2OS细胞中RAG1A的靶向与脱靶位点之间的脱靶染色体易位偏倚。来自三个重复的平均值±SD。虚线表示比率＝1。注意，由于在这些细胞系中相对较少的脱靶染色体易位接合，来自所有已鉴定的脱靶位点的染色体易位接合进行了组合以进行分析。B.示意图显示了利用两个反向引物建立的PEM-seq文库(左图)和使用反向引物PEM-seq文库检测到的脱靶偏倚(右图)。Cas9可以在含sgRNA的断裂末端处持续存在。箭头表示PEM-seq的诱饵引物。C.通过FACS用BrdU和7-AAD双重染色对K562循环细胞和G1停滞的细胞进行细胞周期分析。黑色门表示G1时期。D和E.PEM-seq检测K562正常细胞周期的细胞和G1时期细胞中靶向HBA1，C-MYC1和C-MYC2位点的Cas9的缺失率(D)和编辑效率(E)。F.PEM-seq检测在K562正常细胞周期的细胞和G1细胞中靶向C-MYC1和C-MYC2的Cas9的脱靶位点的染色体易位百分比。倍数变化位于条形的顶部。
图11：A.PEM-seq检测到的HEK293T细胞中HBA1位点的Cas9，Cas9-T2A-TREX2，Cas9X2和Cas9X2d脱靶偏倚。在底部显示已鉴定的脱靶的接合数和计算出的偏差并显示编辑事件的总数。红色数字突出显示了接合很少的位点。B.Circos图显示了在HEK293T细胞中，PEM-seq测定，从C-MYC2克隆的全基因组染色体易位的分布图，例如图3的E图中所述。C-E.在HEK293T细胞中PEM-seq测定，从C-MYC2克隆的Cas9，Cas9-T2A-TREX2，Cas9X2和Cas9X2d的编辑效率(C)，脱靶染色体易位百分比(D)和脱靶偏倚(E)。F和G.与PET-seq检测到的HEK293T细胞中的Cas9相比，Cas9-T2A-TREX2，Cas9X2和Cas9X2d的脱靶染色体易位(F)和一般染色体易位(G)的倍数变化。n＝10，使用的基因座是DNMT1-1，DNMT1-2，EMX1，HBA1，C-MYC1，C-MYC2，C-MYC3，RAG1A，RAG1B和RAG1C。注意，Cas9X2的几个基因座中未检测到脱靶接合。
图12：A.SDS PAGE显示纯化的TREX2，TREX2-3R，Cas9X2和Cas9TX。B.通过体外切割测定法测定，TREX2和TREX2-3R消化38聚体寡核苷酸。C和D.通过PEM-seq检测到的HEK293T 细胞中HBA1(C)或C-MYC2(D)的脱靶位点处的Cas9，Cas9X2和Cas9TX的脱靶偏倚。浅色数字突出显示了接合很少的位点。
图13：A.PEM-seq检测RAG1C基因座处BE4max，ABEmax，Cas9和Cas9TX的编辑结果。编辑产物的百分比显示在右侧。B.PEM-seq检测BE4max，ABEmax，Cas9，Cas9TX的一般染色体易位，编辑事件和总测序事件的接合数，以及RAG1C基因座处的未切割对照。C.PEM-seq检测到的HEK293T细胞中EMX1，C-MYC2，DNMT1-2，RAG1C和BCL11A处Cas9，Cas9TX，BE4max和ABEmax的一般染色体易位百分比。D.PEM-seq检测到的HEK293T细胞中EMX1和MYC2处Cas9，Cas9TX和BE4max脱靶染色体易位的百分比。E.相对于HEK293T细胞中EMX1，C-MYC2，DNMT1-2，RAG1C和BCL11A的编辑效率标准化的Cas9，Cas9TX，BE4max和ABEmax的一般染色体易位百分比。
图14：A-F.PEM-seq检测，转染后3天，7天和14天的CAR T细胞中Cas9和Cas9TX的PDCD1-TRAC(A)，PDCD1-TRBC(B)，TRBC-TRAC(C)，TRBC-PDCD1(D)，TRBC一般染色体易位(E)，PDCD1一般染色体易位(F)的染色体易位比率。3次重复的平均值±SD，平均值显示在每个点的顶部。两尾t检验，*p<0.05，**p<0.01。(G)。混合后24小时通过FACS检测，以指定比例与CAR-T细胞共培养的CD19+和CD19-K562对照细胞的百分比。CD19+K562细胞带有BFP，CD19-K562细胞带有GFP。
图15：Cas9TX减少染色体易位的模型。A.每一轮Cas9切割后产生完全的重新连接，插入缺失和染色体易位。完全的修复产物可以被Cas9反复切割，插入/缺失和染色体易位的水平在每一轮切割中累积。B.Cas9TX通过增强主要对重复切割具有抗性的插入/缺失来抑制染色体易位，从而减少/抑制完全的重新连接。
具体实施方式
发明人基于PEM-seq(Primer-extension-mediated sequencing,PEM-seq)方法追踪了各种DNA异常染色质修复结构的来源，并最终开发了融合蛋白来消除基因编辑过程中的染色体易位和大片段缺失等异常染色质结构。发明人首先在人类的293T细胞系中测试了本发明的融合蛋白，发现染色体易位和大片段缺失的水平均降低了几倍到数十倍不等。具体而言，为了监测CRISPR-Cas9编辑的T细胞中的染色体易位，发明人采用引物延伸介导的染色体易位测序测定以在编辑后的第3、7和14天灵敏地检测TRAC，TRBC和PDCD1基因处被靶向的T细胞中的染色体易位。发明人在转染后第3天观察到大量染色体易位，而在第14天这些染色体易位仍保有许多。发明人进一步发现，在CRISPR-Cas9编辑的T细胞中高水平的染色体易位是由于在靶位点的反复切割所致。因此，开发了一种新的融合蛋白，可防止重复切割，从而在工程化T细胞中进行多重基因组编辑期间大大抑制染色体易位。而且，与野生型Cas9相比，本发明的融合蛋白在许多被靶向的位置处的编辑能力也得到了增强。值得注意的是，除了在多基因编辑时发挥作用，本发明的融合蛋白在进行单基因编辑时可以提高编辑效率并且在单个靶位点进行基因编辑时消除高水平的染色体易位。
下文提供了本文中使用的定义以便于理解本申请。
如本文中所用，PEM-seq(Primer-extension-mediated sequencing,PEM-seq)方法是一种同时测定CRISPR/Cas9的编辑效率和特异性的高通量测序方法，其结合了线性扩增介导的高通量全基因组易位测序(LAM-HTGTS)与靶向测序技术，可以通过易位捕捉选择性检测CRISPR/Cas9脱靶位点并且通过量化Cas9诱导的不完全DSB修复产物评估编辑效率(Yin,J.,Liu,M.,Liu,Y.,Wu,J.,Gan,T.,Zhang,W.,Li,Y.,Zhou,Y.,and Hu,J.(2019).Optimizing genome editing strategy by primer-extension-mediated sequencing.Cell Discov 5,18.)。在评估CRISPR/Cas9编辑效率方面，PEM-seq比目前使用的检测方法还有两个优势：(1)PEM-seq中的引物延伸和随机分子条形码(random molecular barcode,RMB)消除了其他方法(如T7EI、RFLP、TIDE和靶向测序)中使用的PCR扩增过程中的扩增偏差，和(2)PEM-seq 检测小插入/缺失、大缺失、染色体易位(都是CRISPR/Cas9编辑事件)，而其它方法只检测小插入/缺失。PEM-seq方法包括引物延伸、桥式衔接头连接、PCR扩增和Illumina测序的捕捉。具体地，将生物素引物置于切割位点的特定距离，例如200-bp内，将生物素引物和超声处理的基因组DNA进行重复的退火和变性。将产物纯化并且加热，并且在冰上快速冷却以进行DNA变性，用链霉亲合素珠纯化生物素化的产物。将DNA-珠清洗后，用T4DNA连接酶进行桥式衔接头连接。之后，用I5和I7测序引物对珠-DNA复合物进行巢式PCR。通过大小选择珠回收PCR产物，接着用Illumina P5和P7引物进行PCT标签化。所有PEM-seq文库进行测序(例如2×150bp Hiseq)。
如本文中所用，基因编辑(gene editing)是对生物体的基因组及其转录产物进行定点修饰或者修改以改变目的基因或调控元件的序列、表达量或功能。早期基因编辑技术包括归巢内切酶、锌指核酸内切酶和类转录激活因子效应物。近年来，以CRISPR/Cas9系统为代表的新型技术使基因编辑的研究和应用领域得以迅速拓展。“单基因编辑”是指对生物体的单个基因进行基因编辑。“多基因编辑”是指同时对生物体的多个基因进行基因编辑。
如本文中所用，“CRISPR-Cas”系统是由成簇规律间隔短回文重复序列(CRISPR)和CRISPR结合蛋白(即Cas蛋白)组成的核酸酶系统，能够对真核细胞中几乎所有与原型间隔子邻近基序(protospacer-adjacent motif,PAM)相邻的基因组序列进行切割。一般而言，CRISPR系统的特征为促进在靶序列(在内源CRISPR系统中又称为原型间隔子)的位点处的CRISPR复合物形成的元件。“CRISPR”是指成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats)，该序列是许多原核生物的免疫系统。CRISPR中包含RNA组分，有时被称为引导RNA(gRNA)。引导RNA一般包含引导序列和骨架序列，这两个序列可以在同一个分子中或不同的分子中。引导RNA的作用为引导Cas蛋白切割与引导序列互补的DNA位点，也即靶序列。一般而言，引导序列是与靶序列具有足够互补性以便与该靶序列杂交、并且引导CRISPR复合物与该靶序列特异性结合的任何多核苷酸序列。引导序列与其相应的靶序列之间的互补程度是约或多于约50％或更多。一般而言，引导序列的长度为约或多于约12个核苷酸。骨架序列为引导RNA中必须的，除引导序列之外的其余序列，一般包含tracr序列和tracr配对序列，这些序列一般不会因为靶序列的变化而改变。引导RNA包括单链引导RNA(sgRNA)以及由crRNA和tracrRNA组成的双链引导RNA。在本文中，引导RNA可以是单链引导RNA(sgRNA)。
在CRISPR复合物形成的背景下，“靶序列”是指设计具有互补性的引导序列所针对的目标序列，其中在靶序列与引导序列之间的杂交促进CRISPR复合物的形成。完全互补性不是必需的，条件是存在足够互补性以引起杂交并且促进CRISPR复合物的形成。CRISPR复合物的靶多核苷酸可以是对真核细胞而言内源或外源的任何多核苷酸。例如，该靶多核苷酸可以是驻留在真核细胞的细胞核中的多核苷酸。靶多核苷酸可以是编码基因产物(例如，蛋白质)的序列或非编码序列(例如，调节多核苷酸或无用DNA)。不希望被理论所束缚，该靶序列应当与PAM(原型间隔子邻近基序)相关；也就是说，由CRISPR复合物识别的短序列相关。在本文中，靶序列可以是选自下组的基因：Oct4、MYH7、TRAC、PDCD1、TRBC、UROS、Fzd3、TLX1(HOX11)、TLX3(HOX11L2)、HOXA簇、HAL1、HAL2、LYL1、BHLHB1、LMO1、LMO2、LCK、NOTCH1、CCND2、SIL/TAL1、CALM/AF10、MLL/ENL、MLL/AF6、MLL/AF10、MLL/AFX1、MLL/AF4、NUP214/ABL1、EML1/ABL1、ETV6(TEL)/ABL1、ETV6(TEL)/JAK2、BCR/ABL1、NUP98/RAP1GDS1、B2M、CCR5或VEGFA。
如本文中所用，术语“Cas蛋白”是指CRISPR相关蛋白。Cas蛋白的非限制性实例包括：Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8，Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、以及新近发现的Cas12e、其同系物、或其修饰形式。
如本文中所用，“Cas9蛋白”，也称为Csn1，是既参与crRNA生物合成又参与摧毁入侵DNA的蛋白质。已经在不同的细菌物种如嗜热链球菌(S.thermophiles)、无害利斯特氏菌(Listeria innocua)和化脓性链球菌(S.Pyogenes)中描述了Cas9。Cas9蛋白含有两个预测的核酸酶结构域，即位于蛋白质中部的HNH(McrA样)核酸酶结构域和分裂的RuvC样核酸酶结构域(RNAase H折叠)。Cas9变体可以是不天然存在于自然界中并且是由蛋白质工程或通过随机诱变获得的Cas9核酸内切酶。例如，可以通过突变，即化脓性链球菌Cas9核酸内切酶的氨基酸序列中至少一个残基的缺失或插入或取代获得Cas9变体。在一些实施方案中，Cas9蛋白是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9，并且可包括源自于这些生物体的突变的Cas9。这些Cas9是已知的。例如，化脓链球菌(Streptococcus pyogenes)Cas9蛋白的氨基酸序列可见于SwissProt数据库登录号Q99ZW2下，脑膜炎奈瑟氏菌(Neisseria meningitides)Cas9蛋白的氨基酸序列可见于UniProt数据库编号A1IQ68，嗜热链球菌(Streptococcus thermophilus)Cas9蛋白的氨基酸序列可见于UniProt数据库编号Q03LF7，金黄色葡萄球菌(Staphylococcus aureus)Cas9蛋白的氨基酸序列可见于UniProt数据库编号J7RUA5。在本文中，Cas9蛋白可以包含SEQ ID NO:2的氨基酸序列或者可以包含SEQ ID NO.2的氨基酸序列进行了添加、缺失、取代或插入的序列，只要得到的序列具有Cas9蛋白的天然功能。
如本文中所用，“Cas12a蛋白”，又称为Cpf1，是一种缺乏小反式编码RNA的单链RNA引导的内切核酸酶。Cas12a蛋白使用富含T的原型间隔子邻近基序，该基序包含2-6个碱基对的DNA序列以及后面的由CRISPR细菌适应性免疫系统中的Cas9核酸酶靶向的DNA序列。Cas12a蛋白识别富含T的PAM,TTTN，但是该序列是在引导物的5'侧。Cas12a蛋白通过staggered DNA双链断裂切割DNA。Cas12a蛋白最近已经被用作可选的基因组编辑工作，作为一种在基因编辑中有用的分子剪刀。相比于Cas9系统，Cas12a的编辑效率与Cas9的效率相当，在有些靶点低于Cas9。Cas12a的脱靶率极低，相比于Cas9脱靶率高的特性，Cas12a是一种安全的基因编辑工具。Cas12a在切割之后形成粘性末端，而Cas9形成平末端，已有研究表明，Cas12a切割之后的粘性末端相比于Cas9的平末端而言，更容易发生同源重组修复，这也为基因的定点插入和修复提供了更好的工具。在引导RNA的加工方面，Cas12a具有明显的优势，仅仅只需要Cas12a本身就能够完成对前提RNA的加工，而Cas9系统则需要RNaseIII的加工，这极大地促进Cas12a在多基因编辑上的应用。在PAM的识别上，Cas12a识别5’-TTTN-3’或5’-KYTV-3’，Cas9则识别5’-NGG-3’。在本文中，Cas蛋白可以是Cas12a蛋白，例如AsCas12a蛋白。Cas12a蛋白可以包含SEQ ID NO.91的氨基酸序列或由包含SEQ ID NO.92的核酸编码。
如本文中所用，“Cas12e蛋白”是通过对地下水细菌的宏基因组分析鉴定，并表征为RNA引导的DNA核酸酶的Cas蛋白(Liu,J.J.et al.Nature 566,218–223(2019)。它识别5’-TTCN PAM，并在呈递sgRNA(共价连接的crRNA-tracrRNA)时能够在大肠杆菌中进行质粒干扰。除了位于C末端的RuvC域外，它与其他报道的Cas核酸内切酶没有相似之处。Cas12e的上述特征与Cas12的特征相关；然而，Cas12e(约980aa)的大小小于报道的Cas12的大小(约1200aa)。在Liu等人的论文中发现结合sgRNA的Deltaproteobacteria Cas12e(DpbCas12e)包含20-nt引导片段并识别TTCN PAM元件，导致具有10-nt交错末端的dsDNA靶标切割。据报道，当通过与crRNA引导互补ssDNA结合触发时，Cas12表现出不依赖PAM的非特异性ssDNA反式切割活性。Liu等人(Burstein,D.et al.Nature 542,237–241(2017)和Swarts,D.C.&Jinek,M.Mol.Cell 73,589–600.e4(2019))发现Cas12e的反式ssDNA切割活性显著低于Cas12a和Cas12b的活性。他们进一步研究了Cas12e在大肠杆菌和人类细胞中进行基因编辑的潜力。他们发现DpbCas12e能够切割大肠杆菌细胞中的靶基因，而其失活的对应物能够结合靶基因并减少基因表达。此外，他们还表明相关的浮霉菌(Planctomycetes)Cas12e(PlmCas12e)在人类HEK293T细胞中具有基因编辑能力。所有这些数据都为Cas12e作为一种新的基因编辑和CRISPRi工具的发展提供了新的见解。在本文中，Cas蛋白可以是Cas12e蛋白，例如PlmCas12e蛋白。Cas12e蛋白可以 包含SEQ ID NO.89所示的氨基酸序列或者可以以包含SEQ ID NO.90的核酸编码。
如本文中所用，“TREX2蛋白”指具有3’至5’外切核酸酶活性并且消除错配的、修饰的、片段化的和正常的核苷酸以产生适合于DNA代谢途径的后续步骤的3’末端的蛋白质。TREX2在DNA复制、修复和重组中发挥作用。在本文中，Trex2蛋白可以包含SEQ ID NO.1的氨基酸序列或者可以包含SEQ ID NO.1的氨基酸序列进行了添加、缺失、取代或插入的序列，只要得到的序列具有本文所述的Trex2蛋白的功能，例如该Trex2蛋白保留消除DNA结合活性的163A，165A和167A的氨基酸残基，或者保留其酶促活性的氨基酸残基。在本文中，可以通过将Cas蛋白，例如Cas9与TREX2的突变体(含有R163A、R165A和R167A中的一个或多个)直接偶联或用接头偶联生成融合蛋白。Trex2蛋白可以是野生型Trex2蛋白(SEQ ID NO.8)或其中引入R163A、R165A和R167A中的一个或多个的变体。
如本文中所用，“变体”是指通过在亲本分子的氨基酸序列中突变或取代至少一个残基获得的分子，该分子保留亲本分子的功能特征。
如本文中所用，“TALE核酸酶”或“TALEN”意指由通常源自转录活化因子样效应物(TALE)的核酸结合结构域和一个核酸酶催化域组成切割核酸靶序列的融合蛋白。催化域优选是核酸酶结构域，更优选具有核酸内切酶活性的结构域，例如I-TevI、ColE7、NucA和Fok-1。在特定实施方案中，TALE结构域可以与大范围核酸酶融合，例如I-CreI和I-OnuI或其功能变体。在更优选的实施方案中，核酸酶是单体TALE核酸酶。单体TALE核酸酶是不需要二聚化以进行特异性识别和切割的TALE核酸酶，例如WO2012138927中所述的工程化的TAL重复序列与I-TevI的催化结构域的融合体。转录活化子如效应物(TALE)来自细菌物种中黄单胞菌属的蛋白质，包含多个重复序列，每个重复序列在位置12和13上包含特异于核酸靶向序列中每个核苷酸碱基的两个残基(RVD)。具有相似的模块化碱基对碱基核酸结合特性(MBBBD)的结合结构域也可以源自最近在不同细菌物种中发现的新模块化蛋白质。新的模块化蛋白具有比TAL重复序列显示更多序列变异性的优势。优选地，与识别不同核苷酸相关的RVD为：识别C的HD，识别T的NG，识别A的NI，识别G或A的NN，识别A、C、G或T的NS，识别T的HG，识别T的IG，识别G的NK，识别C的HA，识别C的ND，识别C的HI，识别G的HN，识别G的NA，识别G或A的SN，识别T的YG，识别A的TL，识别A或G的VT和识别A的SW。TALE核酸酶已经被描述并用于刺激基因靶向和基因修饰(Boch,Scholze等.2009；Moscou和Bogdanove 2009；Christian,Cermak等.2010；Li,Huang等.2011)。定制的TAL核酸酶可以商品名TALENTM商购获得(Cellectis,8 rue de la Croix Jarry,75013 Paris,France)。在本文中，第一蛋白可以是任何合适的TALEN。
如本文中所用，“锌指核酸酶”或“ZFN”由一个DNA识别域和一个非特异性核酸内切酶构成。DNA识别域是由一系列Cys2-His2锌指蛋白串联组成，一般3～4个。每个锌指蛋白识别并结合一个特异的三联体碱基。锌指蛋白源自转录调控因子家族，在真核生物中从酵母到人类广泛存在，形成alpha-beta-beta二级结构。其中alpha螺旋的16氨基酸残基决定锌指的DNA结合特异性，骨架结构保守。对决定DNA结合特异性的氨基酸引入序列的改变可以获得新的DNA结合特异性。现已公布的从自然界筛选的和人工突变的具有高特异性的锌指蛋白可以识别所有的GNN和ANN以及部分CNN和TNN三联体。多个锌指蛋白可以串联起来形成一个锌指蛋白组识别一段特异的碱基序列，具有很强的特异性和可塑性，适合用于设计ZFN。与锌指蛋白组相连的非特异性核酸内切酶来自FokI的C端的96个氨基酸残基组成的DNA剪切域(Kim et al.,1996)。FokI是来自海床黄杆菌的一种限制性内切酶，只在二聚体状态时才有酶切活性(Kim et al.,1994)。每个FokI单体与一个锌指蛋白组相连构成一个ZFN，识别特定的位点，当两个识别位点相距恰当的距离(6～8bp)时，两个单体ZFN相互作用产生酶切功能，从而达到DNA定点剪切的目的。在本文中，第一蛋白质可以是任何合适的ZFN。
如本文中所用，“接头”指连接两个蛋白部分的一个或多个氨基酸残基。接头可以是柔性接头。在本文中，接头是(G)n、(GGGGS)n、(EAAAK)n、(XP)n或XTEN接头，其中n是大于等于1的整数， X是任何氨基酸，优选丙氨酸、赖氨酸或谷氨酸。优选地，接头是(G)4S接头。本领域技术人员可以选择不同的接头来连接两个蛋白部分，例如Trex2蛋白与Cas9蛋白。n可以是2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90或100，或其间的任何整数。
如本文中所用，“融合蛋白”指两个以上的蛋白部分连接在一起形成的蛋白质，其中蛋白部分各自能够发挥它们的功能。蛋白部分之间可以是直接连接的或者可以通过接头连接，只要蛋白部分各自能够发挥它们的功能。本发明的融合蛋白包含第一蛋白和第二蛋白。第一蛋白是Cas蛋白，TALEN或ZFN，第二蛋白是核酸外切酶或者核酸内切酶。例如，本发明提供了融合蛋白Cas9TX，其是目前基因编辑领域第一个主要用于降低基因编辑过程中的异常染色质修复结构的基因编辑酶。基因编辑酶Cas9TX是通过将Cas9与TREX2的突变体(R163A、R165A和R167A)用(G)4S接头偶联生成。Cas9TX保证基因编辑过程中的基因组稳定性，降低染色体异常结构的产生并且提高CAR-T构建过程中T细胞的基因组稳定性，防止改造的CAR-T细胞发生瘤变。本发明的融合蛋白使用细胞内源的Trex2蛋白的突变体，在使用上具有安全性，其是目前基因编辑领域第一个主要用于降低基因编辑过程中的异常染色质修复结构的基因编辑酶。在本文中，融合蛋白包含SEQ ID NO.3的氨基酸序列或者可以包含SEQ ID NO.3的氨基酸序列进行了添加、缺失、取代或插入的序列。
如本文中所用，“染色体结构变异”通常是指染色体物质的区段以在野生型或者正常细胞中不存在方式异常连接。染色体结构变异的实例包括染色体缺失、扩增、反转、染色体易位等。在本文中，染色体结构变异可以指染色体缺失和/或染色体易位。
如本文中所用，“染色体易位”通常是指在相同或不同的两个染色体之间染色体物质的等量或非等量交换。
如本文中所用，“染色体缺失”或“染色体片段缺失”是指染色体上100bp-4Mb的染色体片段缺失。染色体的大片段缺失可以是200bp、300bp、400bp、500bp、600bp、700bp、800bp、900bp、1kb、5kb、10kb、20kb、30kb、40kb、50kb、100kb、200kb、300kb、500kb、600kb、700kb、800kb、1Mb、2Mb或3Mb的染色体的大片段缺失。
如本文所用，过继细胞转移是将细胞转移到患者体内。这些细胞可能来自患者本人，随后在转移回之前(自体转移)发生了改变，或者它们可能来自另一个体。细胞最常见地是来源于免疫系统，目的是将改善的免疫功能和特性与细胞一起转移回至患者。转移自体细胞或来自患者的细胞可最大程度地减少移植物抗宿主疾病(GVHD)或者组织或器官排斥。
如本文所用，“嵌合抗原受体(chimeric antigen receptor)”或“CAR”是指可用于将一种或多种抗原结合部分移植到免疫效应细胞(诸如T细胞)的基因工程受体。一些CAR也称为“人工T细胞受体”、“嵌合T细胞受体”或“嵌合免疫受体”。CAR可以包含对一种或多种抗原(诸如肿瘤抗原)具有特异性的胞外抗原结合域、跨膜域和T细胞和/或其他受体的胞内信号传导域。嵌合抗原受体修饰的免疫细胞使用遗传工程手段修饰免疫细胞使其表达外源性CAR基因。CAR基因主要包括细胞外识别域和细胞内信号转导结构域：前者用于识别靶细胞表面特异性分子(例如肿瘤表面特异性分子)，后者用于启动识别肿瘤表面分子后的免疫细胞应答，发挥细胞毒作用。嵌合抗原受体主要以T-细胞为载体，“CAR-T”即指表达CAR的T细胞。
如本文所用，“T细胞受体”或“TCR”指在T细胞表面上存在的分子，其负责识别在抗原呈递细胞(APC)表面上展示的抗原。每个T细胞都表达独特的TCR，其是通过随机分类基因而产生的，从而确保T细胞可以对几乎任何感染做出反应。TCR还能够从细胞内部识别肿瘤特异性蛋白(抗原)。当肿瘤特异性蛋白(即LMP2)分解成片段时，其会与主要组织相容性复合物(MHC)一起出现在细胞表面。MHC I类分子呈递来源于细胞内部蛋白的肽抗原。可以对TCR进行工程化改造以识别肿瘤特异性蛋白片段/MHC组合。TCR的结构式由包含α链和β链的两种不同蛋白链构成的。异二聚体TCR蛋白通常由高度可变的α链和β链组成，表达为与不变的CD3链分子的复合物的一部分。TCR的α链和β链两者的可 变结构域分别具有三个高可变或互补性决定区(CDR)。
如本文所用，术语“TCR T细胞”指已通过分子生物学方法对其进行选择、分离和/或鉴定，以表达响应于目标抗原激活的T细胞受体的T细胞或T细胞群体。T细胞群体可以包含外周血单个核细胞(PBMC)。PBMC可以包含淋巴细胞(T细胞、B细胞、NK细胞)、单核细胞和粒细胞(中性粒细胞、嗜碱性粒细胞和嗜酸性粒细胞)。在人类中，淋巴细胞占PBMC群的大部分，其次是单核细胞，而树突状细胞的百分比很小。可以在包含TCR的任何细胞上修饰TCR，所述细胞包括例如辅助T细胞、细胞毒性T细胞、记忆T细胞、调节性T细胞、自然杀伤T细胞和γδT细胞。
蛋白质变体
在本发明的融合蛋白中，可以对具体的蛋白质进行适当的修改以得到蛋白质变体。例如，可以对本发明的每个蛋白质的氨基酸序列，例如SEQ ID NO:1、2或3所示的氨基酸序列取代、添加、缺失或插入1个或多个，优选2、3、4或5个氨基酸残基的氨基酸序列。
氨基酸添加指在氨基酸序列，例如SEQ ID NO:1或2的C端或N端添加氨基酸。
氨基酸取代指在氨基酸序列，例如SEQ ID NO:1或2的序列的某个位置的某个氨基酸残基被其他氨基酸残基替代。
氨基酸插入指在氨基酸序列例如SEQ ID NO:1或2的序列的适当位置插入氨基酸残基，插入的氨基酸残基也可以全部或部分彼此相邻，或插入的氨基酸之间都不彼此相邻。
氨基酸缺失指可以从氨基酸序列，例如SEQ ID NO:1或2的序列中缺失1、2或3个以上氨基酸。
在本发明中，取代可以是保守氨基酸取代，指与SEQ ID NO:1或2的氨基酸序列相比，有3个，更佳地2个氨基酸或1个氨基酸被性质相似或相近的氨基酸所替换而形成肽。这些保守性变异肽可以根据表1进行氨基酸替换而产生。
在本发明的上下文中，保守取代可以根据以下三个表中的一个或多个中反映的氨基酸类别内的取代来定义：
表1：保守取代的氨基酸残基类别
Figure PCTCN2022110597-appb-000001
表2：备选保守氨基酸残基取代类别
1 A S T
2 D E  
3 N Q  
4 R K  
5 I L M
6 F Y W
表3：氨基酸残基的备选物理和功能分类
Figure PCTCN2022110597-appb-000002
Figure PCTCN2022110597-appb-000003
本发明的蛋白质变体可以通过序列同一性进行表征。为了本发明的目的，使用Needleman-Wunsch算法(Needleman and Wunsch,1970,J.Mol.Biol.48:443-453)测定两个氨基酸序列之间的序列同一性，如在EMBOSS包(EMBOSS:The European Molecular Biology Open Software Suite,Rice等,2000,Trends Genet.16:276-277)，优选第5.0.0版以上的Needle程序中实施。使用的参数是缺口开放罚分10，缺口延伸罚分0.5和EBLOSUM62(BLOSUM62的EMBOSS版本)替代矩阵。标记为“最长同一性”的Needle的输出(使用-nobrief选项获得)用作百分比同一性，并如下计算：
(相同残基×100)/(比对长度-比对中的缺口总数)。
在本文中提及第一蛋白和第二蛋白时，特定的蛋白涵盖野生型蛋白质或与其具有氨基酸序列同一性的蛋白质变体。氨基酸序列同一性可以至少85％、90％、95％、96％、97％、98％或99％氨基酸序列。例如提及TREX2蛋白涵盖SEQ ID NO.8的野生型蛋白氨基酸序列或其中引入R163A、R165A和R167A中一个或多个的氨基酸序列，或者与它们具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
递送方法
可以考虑本领域中任何已知的方法对细胞内递送本发明的蛋白质或组合物。非限制性的实例包括病毒转导，电穿孔转染，脂质体递送，聚合物载体，化学载体，脂质复合物，聚合复合物，树枝状聚合物，纳米粒子，乳剂，天然内吞或吞噬途径，细胞穿透肽，显微注射法，微针递送法，粒子轰击法等。
优选的实施方案为电穿孔转染法，可以使用的电传孔仪器的非限制性实例包括：Neon转染系统(Thermo Fisher Scientific)，Gemini仪器和AgilePulse/CytoPulse仪器(BTX-Harvard apparatus)，4D-Nucleofector系统、Amaxa Nucleofector II、Nucleofector 2b仪器(Lonza)，CTX-1500A仪器(Celetrix)，MaxCyte GT或VLX仪器(MaxCyte)，Gene Pulser Xcell(Biorad)。在厂商的指导的基础上，可修改脉冲持续时间、强度，脉冲之间的间隔，脉冲次数，已达到高转染效率而低死亡率的最佳条件。在本发明的实施方案中，主要转染的细胞类型为人类原代的T细胞、淋巴细胞、外周血单个核细胞，理论上多数细胞类型都可以使用电穿孔转染方法将载体递送至细胞内。
细胞
本发明的CRISPR-Cas系统能用于制备表达嵌合抗原受体(CAR)或T细胞受体的细胞。根据本发明要进行修饰的细胞可以是任何合适的T细胞。例如，T细胞可以是炎性T淋巴细胞、细胞毒性T淋巴细胞、调节性T细胞或辅助性T淋巴细胞。具体而言，T细胞是细胞毒性T淋巴细胞。在某些实施方案中，T细胞选自CD4+T淋巴细胞和CD8+T淋巴细胞。它们能够从血液中提取或源自干细胞。干细胞可以是成年干细胞、胚胎干细胞，更具体而言是非人类干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞。典型的人类细胞是CD34+细胞。在具体实施方案中，根据本发明要进行修饰的T细胞是人类T细胞。在扩增和遗传修饰本发明的细胞之前，可以通过各种非限制性方法从受试者，如患者获得细胞来源。T细胞可以获自许多非限制性来源，包括外周 血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、感染位点的组织、腹水、胸腔积液、脾脏组织和肿瘤。在本发明的某些实施方案中，可以使用本领域技术人员可利用的和已知的任何数量的T细胞系。在另一个实施方案中，细胞可以源自健康供体或源自确诊患有疾病的患者。在另一实施方案中，细胞是存在不同表型特性的细胞的混合群体的一部分。
核酸和宿主
本发明还提供了TREX2蛋白、Cas9蛋白和融合蛋白的编码核酸。本领域技术人员可以使用核酸来表达这些蛋白质。例如，可以在重组细胞中表达本文提及的各种蛋白质。细胞的类型不受限制，例如细胞可以是真核细胞或者原核细胞。真核细胞可以是真菌细胞，例如酵母细胞，或者昆虫细胞或哺乳动物细胞，例如小鼠细胞。原核细胞可以是细菌细胞，例如大肠杆菌细胞。蛋白质的核酸可以随使用的宿主细胞进行密码子优化。该核酸可以克隆到合适的表达载体中，然后将表达载体导入宿主细胞以进行表达。表达载体的类型不受限制，并且是本领域技术人员公知的。
给出以上描述是为了使本领域技术人员能够制造和使用本发明，并且在特定应用及其要求的背景下提供的。对优选实施方案的各种修改对于本领域技术人员而言将是显而易见的，并且在不脱离本发明的精神和范围的情况下，本文中定义的一般原理可以应用于其他实施方案和应用。因此，本发明无意限制于所示的实施方案，而是与本文公开的原理和特征一致的最宽范围相一致。
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方案的限制。本发明的范围由所附权利要求书限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改和改变也包含在本发明的范围内。
材料和方法
T细胞和电穿孔
使用阴性选择试剂盒(STEMCELL Technologies，目录号19051)从人脐带血中获得人原代T细胞。在37℃和5％CO 2下在RPMI 1640(Corning)中培养T细胞，RPMI 1640含有30单位/ml重组人白介素2(IL-2，Gibco，目录号PHC0021)，谷氨酰胺(Corning)，15％胎牛血清(FBS，Excell Bio)，以及青霉素/链霉素(Corning)。在转染Cas9 RNP之前，以1：1比例的人类抗CD3/28珠激活T细胞3天(Thermo Fisher，目录号11161D)。对于电穿孔，将9μg Cas9/Cas9TX和6μg sgRNA(对于TRAC，TRBC和PDCD1每个位点分别为2μg，在GeneScript中合成了2'-O-甲基和硫代磷酸酯修饰的sgRNA)在室温混合15分钟，然后使用Celetrix电穿孔仪将其转染到100万个T细胞中，电穿孔仪的参数为Vset＝420V,Tset＝20ms,Pnum＝1N,Tint＝1ms。在无磁珠刺激的情况下在培养物中回收T细胞1天，然后再加入抗CD3/28的磁珠进行进一步实验。使用PE/Cyanine7抗人TCRα/β(BioLegend，目录号306719)检查TCR敲除效率。
慢病毒包装和人T细胞感染
使用PEI(Sigma，P3143)将5μg pMD2.G(Addgene 12259),10μg psPAX2(Addgene 12260)和20μg抗CD19 scFV 4-1BB质粒共转染到10cm皿中培养的HEK293T细胞。用0.45μm过滤器过滤上清液，然后使用Beckman Optima L8-80XP在70,000g下于4℃浓缩2小时。用8μg/mL polybrene(Sigma，TR-1003)用慢病毒以500g感染活化的人类T细胞(刺激后1天)2小时。
纯化Cas9，Cas9高保真变体，TREX，TREX-3R，Cas9X2和Cas9TX
使用pET28a主链(Addgene 53135)表达Cas9，Cas9高保真变体Cas9X2和Cas9TX。使用pDB-His-MBP(Addgene 123365)表达TREX2和TREX2-3R突变体。
为了表达MBP-TREX2融合蛋白，将pDB-His-MBP-TREX2质粒转染到大肠杆菌BL21(DE3)Rosetta细胞中。该方法参考先前的方法(Mazur和Perrino，2001)，只是MBP裂解使用TEV代替Genenase进行。
为了表达Cas9，Cas9变体，Cas9X2和Cas9TX，将pET28a质粒转化到大肠杆菌BL21(DE3)Rosetta细胞中，并通过IPTG(Amresco，0487)诱导表达。通过在含有1mM PMSF的裂解缓冲液(20mM HEPES，pH 7.5，10％甘油，0.1％Triton X-100)中超声处理来裂解细胞，并通过在20,000g下离心1小时去除细胞碎片。将上清液加载到HisTrap HP色谱柱(GE Healthcare)上，并在裂解缓冲液中用0至300mM的咪唑梯度洗脱。然后，将组分在Superdex200柱(GE Healthcare)中进行凝胶过滤。最后，使用BSA蛋白质标准品对纯化的蛋白质进行定量，并在使用前储存在-80℃的裂解缓冲液中。
用于TRAC-TRBC，TRAC-PDCD1，TRBC-PDCD1染色体易位的PCR
对于第一轮PCR，GTGTCACAAAGTAAGGATTCTG和CTAGTCTTGTCTGCTACCTGGATC用于TRAC-TRBC染色体易位的扩增，GTGTCACAAAGTAAGGATTCTG和GCACCCTCCCTTCAACCTGACCTGGGAC用于TRAC-PDCD1染色体易位的扩增，CTAGTCTTGTCTGCTACCTGGATC和GCACCCTCCCTTCAACCTGACCTGGGAC用于TRBC-PDCD1染色体易位的扩增。回收PCR产物，并对于TRAC-TRBC染色体易位的扩增使用TTCTGATGTGTATATCACAG和CTAGTCTTGTCTGCTACCTGGATC进行第二轮PCR，对于TRAC-PDCD1染色体易位的扩增使用TTCTGATGTGTATATCACAG和GAGAAGGCGGCACTCTGGTG进行第二轮PCR，对于TRBC-PDCD1染色体易位的扩增使用CTAGTCTTGTCTGCTACCTGGATC和GCTCACCTCCGCCTGAGCAG进行第二轮PCR。
PEM-seq分析
如前所述(Yin等人，2019)制备PEM-seq库。为了用PEM-seq途径更好地分析染色体易位，我们开发了一个新的染色体易位过滤器模块以过滤错误的染色体易位接合(junctions)，包括具有相同随机分子条形码(RMB)的接合和具有高度相似的RMB(<2不匹配)的相同接合序列(Liu等人，2021)。简而言之，PEM-seq可以识别基因组编辑产物：完全的重新连接，插入缺失，染色体易位和其他染色体异常。插入缺失与总识别产物的比率定义为编辑效率。插入缺失定义为缺失(<100bp)和插入(<20bp)。对于碱基编辑器，编辑效率是通过对CRISPResso识别的所有产物进行计数来计算的(>0.2％)。主胞嘧啶或腺嘌呤的编辑频率用作BE4max或ABEmax的“所需”编辑效率，以相对于图13的E图中Cas9和Cas9TX进行的相同编辑效率标准化。
序列与目标位点高度相似(≤8nt错配，考虑sgRNA和PAM序列两者)并且在推测的Cas9切割位点处具有接合的染色体易位热点被认为是脱靶位点。通过排除靶位点附近±20kb和脱靶位点附近±100bp的接合计算出总体染色体易位。
为了计算人T细胞，HEK293T和K562细胞中的脱靶偏倚，对位于脱靶位点±100bp内的接合进行计数。注意，对于图10的A图中的脱靶偏倚的统计量，所有RAG1A脱靶的接合进行组合以计算偏倚，这是因为染色体易位接合数目少。表4-1中列出了用于PEM-seq的引物。
在计算染色体大片段缺失时，我们将PEM-seq结果中缺失大于100bp且小于20kb的测序reads定义为染色体大片段缺失。图8中显示了测定结果。
表4-1：用于PEM-seq文库建立的引物序列以下两个序列完全相同，需要确认。
Figure PCTCN2022110597-appb-000004
Figure PCTCN2022110597-appb-000005
Cas9对DNA片段的体外消化
通常，通过使用T7高效转录试剂盒(TransGen Biotech)在体外转录与支架RNA融合的sgRNA。每个反应中包括100nM浓度的Cas9和300nM RNA。在37℃下在以下条件下消化DNA片段2小时：20mM HEPES(pH 7.5)，5％甘油，100mM KCl，1mM二硫苏糖醇，10mM MgCl2和0.5mM EDTA。
对于DNA底物，使用表4中的引物扩增Cas9：TRAC中靶位点及其脱靶位点。通过重叠PCR产生与TRAC脱靶的两个末端连接的TRAC中靶位点的构建(可再靶向和不可靶向的染色体易位产物)。
表4-2：体外验证反复切割的引物
TRAC-F:GTGTACCAGCTGAGAGACTC
OT-DN-R:GGCATAGCCAATCCATTCAGTGATC
OT-UP-F:TTTAGCATTTACTCAAAAGTCCACAATCCA
TRAC-R:TGGTGGCAATGGATAAGGCC
OT-DN-F:AACTGTGCTAGACATGAGGTACTTCATGTCTTGCATCTGGGTCA
OT-UP-R:AACTGTGCTAGACATGAGGTCTAAGGAGATCATTTTGGAGTTTTAAGATC
将K562细胞阻滞于G1期
K562细胞用5μM palbociclib(PD-0332991)HCl(Selleck,S1116)处理36小时，然后进行Cas9质粒转染，并且在5μM palbociclib中再培养，之后收获细胞。为了进行细胞周期分析，将细胞用50μM BrdU标记60分钟，并用多聚甲醛(PFA)在4℃下固定60分钟，然后将抗BrdU(100x，BD)温育40分钟。然后将细胞用7-AAD(250x，BD)染色20分钟，并通过FACS分析。
质粒构建
表5中列出了所有使用的sgRNA。将Cas9，Cas9X2，Cas9-T2A-TREX2，Cas9X2d，Cas9TX，BE4max(Chengqi Yi博士赠送)，ABEmax(Chengqi Yi博士赠送)构建到具有用P2A-mCherry或CMV驱动的mCherry的pX330主链(Addgene 42230)中。sgRNA表达克隆到具有CMV驱动的GFP代替Cas9的不同pX330骨架中。通过重叠PCR获得TREX2-H188A和TREX2-3R突变体，并通过Sanger测序证实。
表5：用于各基因座的sgRNA
基因座 sgRNA序列
RAG1A GCCTCTTTCCCACCCACCTT GGG
RAG1B GACTTGTTTTCATTGTTCTC AGG
RAG1C GCACCTAACATGATATATTA AGG
DNMT1 TTCCCGGCAGATGTTTACCT TGG
DNMT2 CCCTGCAGTTCCCTAACTGA GGG
C-MYC1 GCTTGGCTATGGGAATAGAA AGG
C-MYC2 GAGTCTGGATCACCTTCTGC TGG
C-MYC3 GTACATGCAGTTCTGCATCT TGG
EMX1 GAGTCCGAGCAGAAGAAGAA GGG
HBA1 GTGCAGAGAAGAGGGTCAGT GGG
PTEN GGTGAGTAGCTGGTTCCCGT GGG
TP53 GACCATTACTCAGCTCTGAG GGG
TRAC TGTGCTAGACATGAGGTCTA TGG
TRBC GGAGAATGACGAGTGGACCC AGG
PDCD1 GGCGCCCTGGCCAGTCGTCT GGG
BCL11A TTTATCACAGGCTCCAGGAA GGG
细胞系和转染
在37℃，5％CO 2将HEK293T细胞培养于Dulbecco改良Eagle培养基(Corning)，该培养基含有谷氨酰胺(Corning)、10％胎牛血清(FBS,Excell Bio)、和青霉素/链霉素(Corning)。在37℃，5％CO2将K562细胞培养于RPMI 1640(Corning)，该培养基含有谷氨酰胺、15％FBS和青霉素/链霉素(Corning)。通过在6-cm皿中用PEI(Sigma)共转染3μg Cas9质粒和3μg sgRNA质粒制备HEK293T细胞的文库。通过在SF缓冲液(Lonza)中用FF120程序使用4D-核转染仪将Cas9质粒(1μg/100万细胞)和GFP质粒(1μg/100万细胞)共导入K562细胞。通过用mCherry和/或GFP进行FACS分选收集所有样品细胞。
核酸外切酶测定-时程反应反应分析
反应测定(10μL)包含20mM Tris-HCl(pH 7.5)，5mM MgCl2、2mM DTT，100μg/ml BSA，7.5nM 38-mer寡核苷酸(Genewiz)和TREX2蛋白或Cas9X2。在室温下温育指定的时间。通过添加30μL乙醇终止反应，并真空干燥。将沉淀物重悬于6μL的1×上样缓冲液中，在95℃下变性5分钟，然后在15％变性聚丙烯酰胺凝胶中分离。
检测γH2AX的免疫荧光
将HEK293T细胞在6孔皿中的载玻片上培养。Cas9/Cas9TX转染或10μM依托泊苷(Sigma，S1225)处理24小时后，将载玻片上的细胞在室温下于4％PFA中固定10分钟，然后进行PBS洗涤。然后将细胞用0.5％TritonX-100透化15分钟。在一抗染色之前，将细胞用3％FBS封闭60分钟。然后将细胞与1：500稀释的抗γH2A.X(磷酸S139)(Abcam，ab2893)在室温温育1小时或者在4℃过夜，然后用0.2％Tween洗涤。然后将细胞用1：500稀释的Alexa 488染色荧光二抗在室温下染色60分钟。然后在室温下使用Hochest 33342(Sigma，B2261)以1mg/mL进行核染色15分钟。最后，将载玻片安装到显微镜载玻片上以进行显微镜分析。图像由尼康A1R高速激光共聚焦显微镜采集，并由ImageJ根据说明进行测量。
CAR-T杀伤测定
将具有CD19和BFP表达的K562细胞或具有FITC(但无CD19)的K562细胞与Cas9/Cas9TX编辑的CAR-T细胞以1：1、5：1、10：1的E：T比共培养24小时。然后通过FACS检测杀伤效率，并通过FlowJo 10.4进行分析。
统计分析
如附图中的图例所示，本文使用了t检验和Wilcoxon检验。数据表示为平均值±SD，p<0.05时认为是显著的。
实施例1：在工程化T细胞中观察到大量染色体易位
为了灵敏地检测CRISPR-Cas9编辑的T细胞中的染色体易位，本实施例采用了引物延伸介导的 测序方法(primer extension-mediated translocation sequencin)进行染色体易位捕获，并在本研究中命名为PEM-seq。通常，本实施例将诱饵引物放置在靶位点的断裂末端之一上以生成PEM-seq文库，该文库可以准确地定量靶位点的两个断裂末端之间的插入/缺失(indel)以及诱饵断裂末端和其他DSB末端之间的染色体易位(图9的A图；Yin et al.,2019；Liu et al.,2021)。插入缺失与总测序读段之比定义为编辑效率，而染色体易位百分比计算为染色体易位与插入缺失加上染色体易位之比。本实施例将T细胞从人脐带血血液中富集并通过抗CD3/CD28激活3天，然后对TRAC，TRBC和PDCD1基因进行CRISPR-Cas9处理，如临床规程NCT03399448(Stadtmauer et al.,2020)所用。纯化Cas9蛋白，并与三个sgRNA混合，以核糖核蛋白复合物(RNP)的形式递送；转染后3、7或14天监测T细胞的编辑效率和染色体易位(图1的A图和图9的B图)。CRISPR-Cas9在T细胞中诱导了实质性的编辑结果，转染后第3天，TRAC的编辑效率达到约51.9％，TRAC的编辑效率达到约44.6％，PDCD1的编辑效率达到47.8％(图1的B图和表6)。值得注意的是，TRBC的引导RNA(sgRNA)在同一个TRBC基因中彼此之间有间隔约9.4kb的两个真正的靶位点，本实施例将它们结合起来进行分析。随着培养时间增加到7或14天，本实施例观察到包含编辑产物的细胞百分比变得越来越低，这可能是由于某些编辑细胞的生长迟缓所致(图1的B图)。
为了全面捕获TRAC，TRBC和PDCD1之间的相互染色体易位，本实施例从所有三个靶位点生成了PEM-seq文库。转染后3天，三个靶向基因之间检测到的染色体易位水平在0.37至1.77％之间变化(图1的C和D图)。以TRAC和TRBC之间的染色体易位为例，以TRAC为诱饵时该水平为0.37％，以TRBC为诱饵时该水平为0.58％(图1的C和D图)。与进一步在靶位点周围使用引物进行巢式PCR进一步验证了这些染色体易位(图9的C图)。还发现了Cas9：TRAC的脱靶位点位于先前报道的染色体X的亚端粒区域(Stadtmauer et al.,2020)。TRAC脱靶和三个靶基因之间的染色体易位水平远低于中靶位点之间的染色体易位水平，其范围为0.001％至0.002％(图1的C图)。与之前的报道(Stadtmauer et al.,2020)相一致，在第7天或第14天的染色体易位分析显示染色体易位水平下降(图1的D图)。但是，染色体易位水平在14天时仍保持在0.1％以上(图1的D图)，这表明当总共使用约1×10 8个工程化T细胞时，至少有1×10 5个含有染色体易位的工程化T细胞注入患者体内。
除了来自其他靶位点或脱靶位点的DSB外，与CRISPR-Cas9诱导的DSB同时发生的基因组内自发产生的DSB也可能与靶DSB一起形成一般染色体易位(Frock et al.,2015)。尽管在不同批次的CRISPR-Cas9处理中水平较低且可能不会复现，但这些一般染色体易位也可以通过PEM-seq捕获。一般染色体易位在整个基因组中广泛分布，在转染后3天时范围为具有不同诱饵的总编辑结果的0.51％至1.96％(图1的E图和图9的D图)。一般染色体易位也与靶位点间染色体易位一样显示出下降(图1的E图)。在总共75,000个鉴定的染色体易位中，CRISPR-Cas9靶向涉及不同癌症途径的数十个基因，并它们与TRAC，TRBC或PDCD1基因融合(图1的F图)。考虑到TRAC和TRBC含有两种强增强子E1和E3，这些一般染色体易位也可能对工程化的T细胞的基因组稳定性构成潜在的威胁。图1的具体数据如表6、表8所示。
表6：T细胞数据(对应于图1的原始数据)
Figure PCTCN2022110597-appb-000006
Figure PCTCN2022110597-appb-000007
Figure PCTCN2022110597-appb-000008
表8(对应于图1)
  WT eSp Fe HF1 Hypa
TRAC          
缺失 62473 143187 179764 127134 147635
插入 46690 81112 111029 77557 78333
染色体易位 1226 2675 2765 1151 2858
重复 72 86 138 56 279
编辑事件 110389 226974 293558 205842 228826
总事件 297743 532667 621249 635241 463287
编辑效率 36.7％ 42.6％ 47.3％ 32.4％ 49.4％
TRBC 611 578 640 642 621
TRBC 261        
PDCD1 226 596 476 395 413
           
TRBC 0.24％ 0.25％ 0.22％ 0.31％ 0.27％
           
PDCD1 0.20％ 0.26％ 0.16％ 0.19％ 0.18％
实施例2：高保真Cas9变体无法抑制染色体易位
目前科学工作者们已经开发了几对高保真Cas9变体来改善CRISPR-Cas9的编辑特异性。因此，本实施例测试了高保真Cas9变体eSpCas9，FeCas9，HF1和Hypa(分别记载于Slaymaker et al.,Yin et al.,2020；Kleinstiver et al.,2016；2016；Chen et al.,2017)是否能抑制工程化T细胞中的染色体易位。本实施例纯化了这些Cas9变体，并将它们与TRAC，TRBC或PDCD1sgRNA混合以通过RNP递送转染T细胞(图9的B图)。本实施例在TRAC基因处使用了诱饵引物来生成PEM-seq文库。对于这些变体，本实施例检测到与野生Cas9相近的切割效率(图9的F图)。针对TRAC的脱靶位点，本实施例仅在转染后3天在野生型Cas9文库中检测到(图9的G图)，此结果表明这些Cas9变体的编辑特异性更高。本实施例在野生型Cas9和Cas9变体的PEM-seq文库中观察到了相近的染色体易位水平(图1的G图)。总的来说，这些数据表明高保真Cas9变体可以抑制Cas9的脱靶活性，但不能有效消除染色体易位。
实施例3：CRISPR-Cas9的重复切割提高DSB的频率
染色体染色体易位的频率通过所涉及的两个DSB的相互作用强度和频率决定(Alt et al.,2013)。由于两个给定DSB之间的相互作用强度相对固定，因此本实施例将注意力转向在染色体易位形成中CRISPR-Cas9诱导的DSB频率。CRISPR-Cas9切割后的修复结果包含完全的修复，插入缺失和染色体易位，其中完全修复的产物类似于未切割的靶位点，可被CRISPR-Cas9反复切割以增加DSB频率(图 2的A图)。由于未切割的靶位点和经过完全修复的产物彼此之间是无法区分的，因此本实施例检查了TRAC及其脱靶位点之间的染色体易位接合分布，以验证CRISPR-Cas9的重复切割。
TRAC脱靶位点在sgRNA主体内具有四个突变，可产生两种类型的染色体易位产物：不可靶向的产物，丢失原间隔子相邻基序(PAM)；可再靶向的产物，其在NGG PAM的N处仅一种突变(图2的B图)。Cas9：TRAC在体外对这些片段的消化表明，CRISPR-Cas9可以将可再靶向的片段与TRAC靶向位点一样有效地裂解，而不可靶向的片段几乎不会被切割(图2的C图)。相应地，PEM-seq鉴定出的染色体易位接合显示出脱靶偏倚，即剩余的可再靶向产物仅是不可靶向产物的四分之一(图2的D图)。与此同时，本实施例也在HEK293T细胞中发现了TRAC位点在该脱靶位点处也展示了相似的脱靶偏倚性(图2的E图)。并且一个新的TRAC的脱靶位点其脱靶偏倚性为2(图2的E图)。与之相似的，在HEK293T细胞中，TRBC和PDCD1也展示了从2.3到3.0的脱靶偏倚性(图2的E图)。本实施例使用PEM-seq重新分析了HEK293T细胞中先前的数据(Yin等人，2019)。在HEK293T细胞中的DNMT1，C-MYC，RAG1基因中的两个位点(RAG1A和RAG1B)以及HCT116，U2OS和K562细胞中的RAG1A位点广泛观察到脱靶偏倚(图2的F图和图10的A以及表7)。在HEK293T细胞的RAG1A位点的总共490个接合处，脱靶偏倚的最高水平达到了约5.6倍(图2的F图以及表7)。这些数据证实了CRISPR-Cas9的重复切割，通常在不同细胞类型中发现。为了排除Cas9在切割后停留在断裂位点对所观测偏倚性的影响(Brinkman et al.,2018)，本实施例在HEK293T细胞中在RAG1A位点从DSB的另外一端建立PEM-seq文库(图10的B,左)。正如引物反转导致了可再靶向端与不可靶向端的转变，观测到了偏倚性的反转(图10的B,右)，说明了所观测到的反转并非由Cas9的停滞所引起的。总之，这些数据验证了各种细胞类型中CRISPR-Cas9的重复切割。图2所示的原始数据如表9所示。
表9(对应于图2的原始数据)
Figure PCTCN2022110597-appb-000009
Figure PCTCN2022110597-appb-000010
表9续
Figure PCTCN2022110597-appb-000011
Figure PCTCN2022110597-appb-000012
表9续
Figure PCTCN2022110597-appb-000013
实施例4：重复的切割导致高水平的染色体易位
为了测试重复切割对染色体易位形成的影响，本实施例在G1阻滞细胞中进行了CRISPR-Cas9编 辑和PEM-seq分析，在这些细胞中，断裂末端的加工受到限制，以促进形成完全修复的产物(Symington and Gautier,2011)。为此，在转染CRISPR-Cas9之前，本实施例用5μM CDK抑制剂Palbociclib将K562细胞阻滞于G1期36小时，该CRISPR-Cas9靶向HBA1或C-MYC基因内的两个位点(C-MYC1和C-MYC2)(图10的C图)。通过PEM-seq,相比于全细胞周期的K562细胞，本实施例观察到基因编辑产物中缺失产物的比例在G1时期中有所减少，这一点证实了在G1时期DSB断裂末端加工的减少(图10的D图)。在全细胞周期的的K562细胞中，三个HBA1脱靶的脱靶偏倚分别为1.6、0.8和1.0，而在G1阻滞细胞中显著增加到2.3、4.6和11.3(图2的G)，这意味着CRISPR-Cas9在G1期具有强大的重复切割能力。关于染色体易位，G1阻滞细胞中HBA1和三个脱靶基因之间的染色体易位水平比循环细胞高6.0、64.0和23倍(图2G和图10的E图)。G1阻滞细胞中两个C-MYC基因座处获得相似的结果(图10的E和F图)。
实施例5：Cas9-TREX2抑制重复切割和染色体易位
与G1期中重复切割增强相反，本实施例将Cas9与核酸外切酶融合在一起以通过促进末端加工来抑制重复切割。为此，本实施例将Cas9在C末端与人3'-5'核酸外切酶TREX2(Cas9X2，从N端到C端为Cas9-(G)4S-TREX2)或带有H188A突变的无核酸酶活性(nuclease-dead TREX2)TREX2(Cas9X2d，从N端到C端为Cas9-(G)4S-nuclease-dead TREX2)融合在一起(图3的A图；Perrino et al.,2005)。还使用自切割的T2A接头(核苷酸序列：GAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA；氨基酸序列：EGRGSLLTCGDVEENPGP)代替了(G)4S接头平行测试了Cas9和TREX2的分离形式(T2A-TREX2，从N端到C端为Cas9-T2A-TREX2)。将包含这些CRISPR基因和sgRNA的质粒转染到HEK293T细胞中进行基因靶向，并在72小时后收获基因组DNA，以生成PEM-seq文库。
关于HEK293T细胞中HBA1位点处的CRISPR编辑，本实施例在T2A-TREX2和Cas9X2文库中检测到的缺失水平高于野生型Cas9的情况，表明共表达的和融合的TREX2增强末端加工(图3中的B图)。在HBA1位点处，Cas9X2和T2A-TREX2均显示出相对较高的编辑效率(76.8％、74.6％与56.9％)，而Cas9X2d的编辑效率低于野生型Cas9(图3中的B图)，这支持如下的观点，即TREX2处理Cas9诱导的断裂末端增强了基因组编辑(Cermak et al.,2017)。此外，与野生型Cas9相比，Cas9X2d和T2A-TREX2降低了三个HBA1脱靶位点处的脱靶偏倚，并且Cas9X2几乎消除了所有三个位点处的脱靶偏倚(图3中的C图和图11中的A图)。因此，所有这三种Cas9衍生酶降低脱靶位点和一般染色体易位水平，对于该脱靶位点和一般染色体易位水平，Cas9X2＜＜T2A-TREX2<Cas9X2d<野生型Cas9(图3中的D和E图)。更进一步对于Cas9X2，HBA1的第二个脱靶位点处的染色体易位水平甚至降低到低于0.004％(图3中的D)。与此同时，本实施例在确定的C-MYC2脱靶位点处获得了类似的发现(图11中的B-E图)。本实施例还测试了HEK293T细胞中的其他八个位点并获得了类似的发现，只是Cas9X2的编辑效率主要高于野生型Cas9(图3中的F-I和表8)。Cas9X2和T2A-TREX2都显示出消除染色体易位的强大能力，但Cas9X2的表现优于T2A-TREX2(图3中的H和I图)。对于所有靶位点，与野生型Cas9相比，Cas9X2的脱靶染色体易位(减少27至77倍)和一般染色体易位(减少2至13倍)减少(图11中的F和G图)。图2和图3中的数据如表10所示。
表10对应图2和图3的数据
Figure PCTCN2022110597-appb-000014
Figure PCTCN2022110597-appb-000015
Figure PCTCN2022110597-appb-000016
实施例6：Cas9TX是用于基因组编辑的更优Cas9X2变体
由于参与DNA修复，TREX2在许多细胞类型中表达，并且TREX2异位表达对细胞存活或细胞 周期没有影响(Certo et al.,2012；Mazur and Perrino,2001)。为了进一步提高Cas9X2的安全性，本实施例生成了具有R163A，R165A和R167A突变(TREX2-3R)的Cas9X2变体，以消除TREX2的DNA结合活性(Perrino et al.,2008)。然后，本实施例纯化了TREX2，TREX2-3R及其与Cas9融合的形式，以进行38-nt寡核苷酸的体外消化测定(图12中的A图)。与TREX2相比，TREX2-3R显示出对寡核苷酸的核酸外切酶活性的大幅降低(图12的B图)。在21分钟时0.5nM Cas9X2检测到短于37nt的消化产物，但是对于2.7nM Cas9-TREX2-3R(Cas9TX)在63分钟时也未检测到消化产物(图4的A图)，这意味着Cas9TX具有潜在的更好的安全性。
接下来，本实施例将Cas9TX应用于HEK293T细胞中的基因组编辑，并进行了PEM-seq分析。尽管丢失了TREX2的DNA结合能力，但Cas9TX仍可以精确地定位到HBA1和C-MYC靶位点，并且在这两个位点处显示出比Cas9显示略高的编辑效率，Cas9X2也是如此(图4的B图)。Cas9X2和Cas9TX都有效消除了脱靶偏倚，减少了总染色体易位，但是Cas9TX表现优于Cas9X2(图4的C图，图12中的C和D图)。本实施例接着测试了HEK293T细胞中的其他12个位点。发现与Cas9相比，Cas9TX在这些位点中显示出显著更高的编辑效率并且诱导小得多的脱靶染色体易位(图4的D和E图和图11；3.2至54.8倍)。Cas9TX显著降低一般染色体易位(图4中的F图，表8；2.3至11.7倍)。总的来说，这些数据表明，Cas9TX可以像Cas9X2一样大大抑制染色体易位，并且与DNA的非特异性接触降低。图4对应的原始数据如表11所示。
表11图4对应的原始数据
Figure PCTCN2022110597-appb-000017
Figure PCTCN2022110597-appb-000018
Figure PCTCN2022110597-appb-000019
Figure PCTCN2022110597-appb-000020
表11续
Figure PCTCN2022110597-appb-000021
表11续
Figure PCTCN2022110597-appb-000022
Figure PCTCN2022110597-appb-000023
实施例7：Cas9TX诱导的染色体易位水平与碱基编辑器诱导相当
胞嘧啶碱基编辑器(CBE)和腺嘌呤碱基编辑器(ABE)系统目前也被开发来诱导靶位点上的点突变从而完成基因编辑(Gaudelli et al.,2017；Komor et al.,2016)。由于CBE或ABE不产生DSB，因此在碱基编辑系统中很少形成染色体易位。为了比较Cas9TX和碱基编辑器之间的染色体易位水平，本实施例应用CRISPR-Cas9、CRISPR-Cas9TX、胞嘧啶碱基编辑器BE4max和腺嘌呤碱基编辑器ABEmax(Koblan et al.,2018)，在HEK293T细胞中靶向EMX1、DNMT1、C-MYC、RAG1、和BCL11A基因内的5个靶位点，然后使用PEM-seq进行染色体易位检测。BE4max可以有效靶向多个胞嘧啶，而ABEmax可以靶向编辑窗口内的多个腺嘌呤，如图5的A图中的RAG1C文库所示。值得注意的是，由于编辑窗口中缺少腺嘌呤，因此ABEmax不能靶向EMX1和C-MYC2位点。相比之下，Cas9诱导大量插入/缺失，在断裂位点附近具有最高的碱基损失。有趣的是，Cas9TX在含有17bp截短sgRNA的断裂末端处显示碱基损失的积累(图5的A图)，这是由于切割后驻留的Cas9TX的末端加工造成的(Brinkman et al.,2018)。通过PEM-seq或CRISPResso评估的测试位点处，BE4max和ABEmax的编辑效率通常低于Cas9和Cas9TX的编辑效率(图5的B图和图13中的A图；Clement et al.,2019)。
Cas9在所有五个位点中诱导大量染色体易位，而Cas9TX显示出非常低的染色体易位水平(图5中的C和D图和图13中的B图)，这与上述结果一致。相比之下，BE4max和ABEmax均在测试位点诱导极低水平的一般染色体易位，但高于背景水平(图5中的B和C图和图13中的B图)。尽管Cas9TX中的染色体易位水平在所有测试位点均低于0.45％，但仍略高于两个碱基编辑器(图5中的C图和图13中的C图)。此外，使用Cas9TX检测到低水平的脱靶染色体易位，但使用碱基编辑器几乎检测不到脱靶染色体易位(图13中的D图)。然而，考虑到碱基编辑器的切割效率较低并且编辑效率计算了多个可靶向碱基，当相对于相同的编辑效率标准化时，Cas9TX和BE4max之间的差距将进一步缩小(图13中的E图)。因此，当达到相同水平的基因破坏时，Cas9TX可以几乎与碱基编辑系统一样好地抑制一般染色体易位。图5对应的原始数据如表12所示。
表12 图5对应的数据
Figure PCTCN2022110597-appb-000024
Figure PCTCN2022110597-appb-000025
表12续
MYC2-OT MYC2-OT MYC2-OT
TX TX TX
43885 159409 370517
1218 2452 4626
264 1169 1606
45825 165217 381133
220859 608434 1042125
20.7％ 27.2％ 36.6％
74.8％ 83.1％ 73.7％
27.7％ 32.7％ 49.6％
0.6％ 0.7％ 0.4％
实施例8：Cas9TX显示出检测不到的非特异性损伤活性
为了进一步测试使用Cas9TX进行基因组编辑的安全性，本实施例通过质粒转染在HEK293T细胞中表达了不含sgRNA的Cas9-P2A-mCherry或Cas9TX-P2A-mCherry。P2A是一种自我切割的肽，可确保表达Cas9或Cas9TX的细胞被mCherry标记。转染后72小时，本实施例通过抗H2AX抗体在细胞中标记了DSB信号。如预期的那样，拓扑异构酶抑制剂依托泊苷诱导了以γH2AX标记的大量DSB(图6的A和B图；Canela等人，2017)。在表达Cas9和Cas9TX的细胞中，DSB的水平没有显著升高(图6的A和B图)，表明在没有sgRNA的情况下，Cas9和Cas9TX均显示出检测不到的非特异性全基因组损伤活性。
接下来，本实施例测试了Cas9TX对其他DSB的影响，通过共表达的AsCas12a和Cas9TX与用于AsCas12a的crRNA进行，靶向在HEK293T细胞中C-MYC基因(C-MYC3)。本实施例在AsCas12a靶位点用诱饵引物进行了PEM-seq分析，发现与Cas9TX共表达比与Cas9共表达时，AsCas12a的编辑效率相对更高(18.8％对21.5％；图6的C图)。此外，与Cas9TX共表达比与Cas9共表达时，AsCas12a：C-MYC3的染色体易位更少(1.5％比2.7％；图6的C图)，这表明Cas9TX还可以提高编辑效率并消除其他共表达编辑酶的染色体易位。在这种情况下，本实施例还在确定的Cas9:C-MYC2脱靶位点处使用了带诱饵引物的PEM-seq，以检查Cas9TX对脱靶位点处切割的影响。与Cas9相比，本实施例在Cas9TX的脱靶位点检测到了更高的编辑频率，但是，两者是接近的且与MYC2靶位点的编辑效率成正比(图6的D图)。而且，与Cas9相比，Cas9TX的MYC2脱靶显示少14.6倍的染色体易位(图6的E图)。本实施例还采用了tracking of indels by decomposition(TIDE)粗略地测量了VEGFA或EMX1位点靶向的HEK293T细胞在多个脱靶位点的编辑频率，其中大多数显示出Cas9TX的编辑频率增加，但也与中靶位点的编辑效率成正比(图6的F图)。这些数据表明，Cas9TX可以以相似的水平增强中靶位点和脱靶位点的切割，但可以有效阻止在任何断裂位点处形成染色体易位。
实施例9：Cas9TX消除了工程化CAR T细胞中的染色体易位
为了测试Cas9TX消除工程化T细胞中染色体易位的能力，本实施例首先将编码CD19-41BB-CAR的逆转录病毒(Kawalekar et al.,2016)转导到活化的T细胞中，然后通过RNP递送应用Cas9或Cas9TX 编辑TRAC，TRBC或PDCD1基因(图7的A图)。对于Cas9和Ca9TX群体，CAR逆转录病毒的转导效率非常接近(图7的B图)。转染后3天，CRISPR-Cas9和CRISPR-Cas9TX均在总培养T细胞的40-55％处产生插入缺失，约一半的T细胞失去TCR(图7的C和D图，表6)。与CRISPR-Cas9编辑的细胞相比，在CRISPR-Cas9TX编辑的T细胞中，TRAC，TRBC或PDCD1靶位点之间的染色体易位大大降低(图7的E和F图)。TRAC-TRBC或TRAC-PDCD1的染色体易位百分比分别从0.28％和0.33％降低到0.03％和0.01％(图7的G和H图)。在转染后3天，Cas9TX编辑的T细胞中三个中靶位点间的其他染色体易位也显示10-30倍的大幅减少(图14的A-D图)。转染后7天或14天，染色体易位率进一步降低，染色体易位发生的频率为2,000至30,000之一(图7的F，G图和图14的A-D图)。此外，在Cas9TX编辑的T细胞中，TRAC脱靶与三个靶位中的任何一个之间均未观察到染色体易位接合(图7的F图)。对于一般染色体易位，与CRISPR-Cas9编辑的T细胞相比，Cas9TX编辑的T细胞中的比例在转染后3天下降3-5倍，并且一般染色体易位在转染后7天或14天进一步降低了(图7的I图，图14的E和F图)。
为了测试Cas9或Cas9TX编辑的CAR T细胞杀死CD19+K562细胞的能力，本实施例将CAR T细胞与CD19+K562细胞以不同的比例混合。系统中还包括与CD19+细胞相等数量的CD19-K562细胞，以进行最终标准化(Kawalekar et al.,2016)。Cas9TX编辑的T细胞显示出与Cas9编辑细胞相似的CAR能力，当与CAR T细胞以1:1混合时，约72％的CD19+K562细胞被裂解，而以1:5混合时几乎达到100％(图7的J图和图14的G图)。这些数据表明，CRISPR-Cas9TX可以有效消除CAR T细胞中的染色体易位，而不会影响其CD19靶向功能。对应图7的原始数据如表7所示。
表7 (对应图7的原始数据)
Figure PCTCN2022110597-appb-000026
Figure PCTCN2022110597-appb-000027
表7续
Figure PCTCN2022110597-appb-000028
Figure PCTCN2022110597-appb-000029
讨论
CRISPR-Cas9诱导的DSB经历不同的DNA修复途径以产生一系列的DNA修复结果。非同源末 端连接(NHEJ)途径将两个完整或经过轻微加工的断裂末端直接连接在一起，以产生完全的重新连接产物或较小的插入/缺失(Betermier et al.,2014)。据估计，CRISPR-Cas9切割后超过50％的修复结果是来自两个相邻靶位点的测序数据的完全重新结合(Brinkman et al.,2018；Guo et al.,2018)。完全的重新连接与靶序列无法区别，并且可以被CRISPR-Cas9重复切割。在每一轮切割过程中，几乎没有DSB能够逃脱对DNA损伤响应的监视，并导致染色体易位。因此，染色体易位在整个基因编辑过程中逐渐增加，并最终达到较高水平(图15的A图)。值得注意的是，染色体易位不仅可以发生在Cas9诱导的DSB之间，而且还可以发生在各种细胞活动过程中产生的一般DSB之间(Alt et al.,2013；Casellas et al.,2016；Nussenzweig and Nussenzweig,2010；Tubbs and Nussenzweig,2017)。
已经在许多类型的CRISPR-Cas9编辑的细胞中广泛观察到染色体易位(Eyquem et al.,2017；Liu et al.,2017；Mandal et al.,2014；Ren et al.,2017；Stadtmauer et al.,2020)。据估计，在CRISPR-Cas9靶向的T细胞中的两个靶基因之间以一定频率发生染色体易位，该频率为从PEM-seq粗略计算出的50-300个编辑过的T细胞之一(图1C)。但是，两个I-SceI靶位点之间的染色体易位频率较低，大约为300-1200个细胞之一(Roukos et al.,2013)，这可能是因为I-SceI诱导的粘性DSB末端更倾向于被加工。一致地，还发现具有4bp粘性末端的CRISPR-Cas12a(或Cpf1)诱导的染色体易位水平低于CRISPR-Cas9(Bothmer et al.,2020)。关于染色体易位，在强增强子或致癌基因上进行基因编辑可能有风险。在这种情况下，已经广泛研究了涉及来自抗原受体基因座或c-Myc的强增强子的染色体易位，以能够驱动正在发育的淋巴细胞中的肿瘤发生(Alt et al.,2013；Lieber,2016；Nussenzweig and Nussenzweig,2010)。除了在这项研究中在工程化T细胞中在TRAC和TRBC中观察到染色体易位外，造血干细胞和祖细胞(HSPC)中在CRISPR-Cas9靶向CCR5或B2M基因期间产生的染色体易位(Liu et al.,2017；Mandal et al.,2014；Ren et al.,2017；Stadtmauer et al.,2020)也对干细胞的基因组完整性构成威胁，并影响循环系统和免疫系统。
TREX2与Cas9的融合允许切割后立即末端加工，因此可以减少完整断裂末端的百分比(图3)。因此，在最初的切割时，很大一部分完全重新连接会转变为插入缺失。在这种情况下，即使没有重复切割，Cas9TX仍可以略微提高编辑效率，并且在单个位点(图4)或多基因组编辑方案(图7)下将染色体易位保持在背景水平上(图15的A和B图)。分开表达的TREX2和Cas9也可以提高切割效率，如先前报道的那样(Cermak et al.,2017)，但是在大多数编辑情况下都无法像Cas9TX一样有效地抑制染色体易位，这可能是由于低效率的及时末端加工引起，这可能会导致完全修复产物的泄漏。此外，Cas9和TREX2的分开形式并未针对RNP传递进行优化，而正如本文所展示的，Cas9TX与RNP相容，并且TREX2-3R的小尺寸(236个氨基酸)也可以将Cas9TX包装成分裂型腺相关病毒(AAV；Chew et al.,2016)中。而且，融合的TREX2应能够提高编辑效率，并抑制其他平末端或3’末端基因编辑酶的染色体易位，因为TREX2更优选这些类型的断裂末端。
尽管由于弱的核酸外切酶活性，野生型TREX2的过表达对细胞没有明显的影响(Certo et al，2012)，但还是在TREX2的DNA结合域上引入了突变，以抑制Cas9TX与DNA的非特异性结合(图4)。已证明非特异性DNA结合结构域上的突变能够提高CRISPR-Cas9或碱基编辑器的编辑特异性(Slaymaker et al.,2016；Zhou et al.,2019)。CRISPR-Cas9TX几乎消除了TRAC，TRBC和PDCD1靶位点之间的染色体易位，对CAR的杀伤力没有可检测的影响，优于CRISPR-Cas9(图7)。与Cas9TX相比，高保真度的Cas9变体尽管具有更高的编辑特异性，却无法抑制易位。对于经常用于基因校正但很少用于基因破坏的碱基编辑器，BE4max和ABEmax仍然能够诱导低水平的染色体易位，因为缺口可以低频率转化为DSB(Tubbs and Nussenzweig,2017)。CRISPR-Cas9TX仅比BE4max或ABEmax诱导更高水平的一般染色体易位(图5C)。值得注意的是，碱基编辑器还可以靶向RNA(Grunewald et al.,2019；Zhou et al.,2019)，而CRISPR-Cas9TX则不存在这种能力。总之，CRISPR-Cas9TX确保与碱基编辑器一样相对良好的基因组完整性。
此外，发明人将本发明的融合蛋白成功应用于改造CAR-T的肿瘤疗法中。利用本发明的融合蛋白敲除CAR-T过程中的TCR和PD-1的编码基因，可以有效提高CAR-T的靶向性以及生存周期。使用Cas9之后，在拟回输的CAR-T中，10万个编辑细胞中存在约300个左右的染色体易位。比较而言，在使用Cas9TX的细胞组中，染色体易位的水平已经降到基线水平，即基本消失。发明人还合作进行了小鼠眼科疾病的模型检测，Cas9中存在的大量染色体易位也在Cas9TX处理的小鼠中消失了。Cas9TX可以显著提高单基因与多基因编辑过程中的基因安全性，在基因编辑的临床应用方面具有较大的应用前景。
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序列表
TREX2突变体TREX2-3R氨基酸序列(SEQ ID NO.1)：
Cas9氨基酸序列(SEQ ID NO.2)：
Figure PCTCN2022110597-appb-000030
Cas9TX氨基酸序列(SEQ ID NO.3):
Figure PCTCN2022110597-appb-000031
TREX2突变体核苷酸序列(SEQ ID NO.4):
Figure PCTCN2022110597-appb-000032
Cas9核苷酸序列(SEQ ID NO.5):
Figure PCTCN2022110597-appb-000033
Figure PCTCN2022110597-appb-000034
Figure PCTCN2022110597-appb-000035
Cas9TX核苷酸序列(SEQ ID NO.6):
Figure PCTCN2022110597-appb-000036
Figure PCTCN2022110597-appb-000037
Figure PCTCN2022110597-appb-000038
TREX2野生型序列
核苷酸序列(SEQ ID NO.7)
Figure PCTCN2022110597-appb-000039
氨基酸序列(SEQ ID NO.8)
Figure PCTCN2022110597-appb-000040
nuclease-dead TREX2序列
核苷酸序列(SEQ ID NO.9)
Figure PCTCN2022110597-appb-000041
Figure PCTCN2022110597-appb-000042
氨基酸序列(SEQ ID NO.10)
AsCas12a DNA序列(SEQ ID NO.92)：
Figure PCTCN2022110597-appb-000043
Figure PCTCN2022110597-appb-000044
AsCas12a氨基酸序列(SEQ ID NO.91)：
Figure PCTCN2022110597-appb-000045
PlmCas12e DNA序列(SEQ ID NO.90)：
Figure PCTCN2022110597-appb-000046
Figure PCTCN2022110597-appb-000047
PlmCas12e氨基酸序列(SEQ ID NO.89)：
Figure PCTCN2022110597-appb-000048
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2022110597-appb-000049
Figure PCTCN2022110597-appb-000050
Figure PCTCN2022110597-appb-000051
Figure PCTCN2022110597-appb-000052
Figure PCTCN2022110597-appb-000053
Figure PCTCN2022110597-appb-000054
Figure PCTCN2022110597-appb-000055
Figure PCTCN2022110597-appb-000056
Figure PCTCN2022110597-appb-000057
Figure PCTCN2022110597-appb-000058
Figure PCTCN2022110597-appb-000059
Figure PCTCN2022110597-appb-000060
Figure PCTCN2022110597-appb-000061
Figure PCTCN2022110597-appb-000062
Figure PCTCN2022110597-appb-000063
Figure PCTCN2022110597-appb-000064
Figure PCTCN2022110597-appb-000065
Figure PCTCN2022110597-appb-000066
Figure PCTCN2022110597-appb-000067
Figure PCTCN2022110597-appb-000068
Figure PCTCN2022110597-appb-000069
Figure PCTCN2022110597-appb-000070
Figure PCTCN2022110597-appb-000071
Figure PCTCN2022110597-appb-000072
Figure PCTCN2022110597-appb-000073
Figure PCTCN2022110597-appb-000074
Figure PCTCN2022110597-appb-000075
Figure PCTCN2022110597-appb-000076
Figure PCTCN2022110597-appb-000077
Figure PCTCN2022110597-appb-000078
Figure PCTCN2022110597-appb-000079
Figure PCTCN2022110597-appb-000080

Claims (17)

  1. 融合蛋白，该融合蛋白包含第一蛋白和第二蛋白，
    其中第一蛋白是Cas蛋白，TALEN或ZFN，第二蛋白是核酸外切酶或者核酸内切酶，其中第一蛋白与第二蛋白直接连接或者通过接头连接；
    优选地，所述Cas蛋白选自Cas9蛋白、Cas12a蛋白、Cas12b蛋白或Cas12e蛋白或其变体，所述Cas9蛋白例如SpCas9或SaCas9蛋白，所述Cas12a蛋白例如AsCas12a蛋白，所述Cas12e蛋白例如PlmCas12e；
    优选地，所述核酸外切酶选自TREX2蛋白、TREX1蛋白、APE1蛋白、Artemis蛋白、CtIP蛋白、Exo1蛋白、Mre11蛋白、RAD1蛋白、RAD9蛋白、Tp53蛋白、WRN蛋白、外切核酸酶V、T5外切核酸酶或T7外切核酸酶或其变体；
    优选地，其中所述接头是柔性接头，例如(G)n、(GGGGS)n、(EAAAK)n、(XP)n或XTEN接头，其中n是大于等于1的整数，X是任何氨基酸，优选丙氨酸、赖氨酸或谷氨酸；
    优选地，第一蛋白在融合蛋白的N端或C端；
    优选地，TREX2蛋白包含SEQ ID NO.8的氨基酸序列或其中引入R163A、R165A和R167A中一个或多个的氨基酸序列，或者与它们具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  2. 根据权利要求1所述的融合蛋白，其中TREX2蛋白包含：
    SEQ ID NO.1的氨基酸序列；或SEQ ID NO.1的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或与SEQ ID NO.1的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  3. 根据权利要求1或2所述的融合蛋白，其中Cas9蛋白包含：
    SEQ ID NO.2的氨基酸序列；或SEQ ID NO.2的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或与SEQ ID NO.2的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；
    Cas12e蛋白包含：
    SEQ ID NO.89的氨基酸序列；或SEQ ID NO.89的氨基酸序列中添加、 缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或与SEQ ID NO.89的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；
    Cas12a蛋白包含：
    SEQ ID NO.91的氨基酸序列；或SEQ ID NO.91的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或与SEQ ID NO.91的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的融合蛋白，其包含
    SEQ ID NO.3的氨基酸序列；或SEQ ID NO.3的氨基酸序列中添加、缺失、取代或插入一个或多个氨基酸残基的氨基酸序列；或与SEQ ID NO.3的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  5. 核酸，该核酸编码权利要求1-4中任一项所述的融合蛋白。
  6. 载体，该载体包含权利要求5所述的核酸。
  7. 宿主细胞，该宿主细胞包含权利要求6所述的载体。
  8. 组合物，该组合物包含权利要求1-4中任一项所述的融合蛋白和sgRNA，优选地，其中所述组合物是核糖核蛋白复合物，优选是CRISPR-Cas系统，优选是CRISPR-Cas9、CRISPR-Cas12a、CRISPR-Cas12b或CRISPR-Cas12e系统。
  9. 一种遗传修饰细胞的方法，所述方法包括将权利要求1-4中任一项所述的融合蛋白、权利要求5所述的核酸、权利要求6所述的载体、权利要求8所述的组合物导入细胞的步骤，优选地，其中所述细胞是T细胞、NK细胞或HSC细胞，优选地，其中所述细胞是TCR T细胞或CAR T细胞。
  10. 一种减少或者消除利用CRSPR-Cas进行基因编辑时产生的染色体结构变异的方法，其包括将权利要求1-4中任一项所述的融合蛋白、权利要求5所述的核酸、权利要求6所述的载体或者权利要求8所述的组合物导入目标细胞的步骤，优选地，其中所述细胞是T细胞或HSC细胞，优选地，其中所述细胞是TCR-T细胞或CAR-T细胞。
  11. 一种基因编辑方法，其包括将权利要求1-4中任一项所述的融合蛋白、权利要求5所述的核酸、权利要求6所述的载体或者权利要求8所述的组合物导入目标细胞的步骤，优选地，其中所述细胞是T细胞或HSC细胞，优选地，其中所述细胞是TCR-T细胞或CAR-T细胞。
  12. 权利要求9-11中任一项的方法，其中组合物能够减少或消除基因编辑过程中的染色体结构变异；优选地，所述基因编辑是单基因编辑或多重基因编辑；
    优选地，其中基因选自Oct4、MYH7、TRAC、PDCD1、TRBC、UROS、Fzd3、TLX1(HOX11)、TLX3(HOX11L2)、HOXA簇、HAL1、HAL2、LYL1、BHLHB1、LMO1、LMO2、LCK、NOTCH1、CCND2、SIL/TAL1、CALM/AF10、MLL/ENL、MLL/AF6、MLL/AF10、MLL/AFX1、MLL/AF4、NUP214/ABL1、EML1/ABL1、ETV6(TEL)/ABL1、ETV6(TEL)/JAK2、BCR/ABL1、NUP98/RAP1GDS1、B2M、CCR5或VEGFA；
    优选地，其中染色体结构变异是染色体易位和/或染色体缺失；优选地，其中染色体缺失是100bp-4Mb的染色体片段缺失；优选地，所述基因编辑通过CRISPR-Cas系统进行，优选地CRISPR-Cas系统是CRISPR-Cas9系统、CRISPR-Cas12a、CRISPR-Cas12b或者CRISPR-Cas12e系统。
  13. 细胞，其已经通过权利要求9-12中任一项所述的方法进行了修饰或基因编辑，优选地，其中所述细胞是T细胞或HSC细胞，优选地，其中所述细胞是TCRT细胞、NK细胞或CAR T细胞。
  14. 权利要求13的细胞在制备药物中的用途，所述药物用于治疗需要该细胞的受试者中的疾病；优选地，其中所述疾病是遗传疾病，例如癌症、自身免疫疾病或眼病，例如，杜氏肌营养不良症、β-地中海贫血、镰状细胞贫血、膀胱癌、髓母细胞瘤、子宫颈癌、乙型肝炎、EBV感染、HIV感染和/或隐孢子虫感染、脉络膜血管增生或黑曚症。
  15. 权利要求1-4中任一项所述的融合蛋白、权利要求5的核酸、权利要求6的载体或权利要求8所述的组合物减少或消除基因编辑过程中的染色体结构变异的用途，优选地，其中基因选自Oct4、MYH7、TRAC、PDCD1、TRBC、UROS、Fzd3、TLX1(HOX11)、TLX3(HOX11L2)、HOXA簇、HAL1、 HAL2、LYL1、BHLHB1、LMO1、LMO2、LCK、NOTCH1、CCND2、SIL/TAL1、CALM/AF10、MLL/ENL、MLL/AF6、MLL/AF10、MLL/AFX1、MLL/AF4、NUP214/ABL1、EML1/ABL1、ETV6(TEL)/ABL1、ETV6(TEL)/JAK2、BCR/ABL1、NUP98/RAP1GDS1、B2M、CCR5或VEGFA；
    优选地，其中染色体结构变异是染色体易位和/或染色体缺失；优选地，其中染色体缺失是100bp-4Mb缺失的染色体片段缺失；优选地，所述基因编辑通过CRISPR-Cas系统，例如CRISPR-Cas9系统、CRISPR-Cas12a、CRISPR-Cas12b或者CRISPR-Cas12e进行；优选地，所述基因编辑是单基因编辑或多重基因编辑。
  16. 权利要求1-4中任一项所述的融合蛋白、权利要求5的核酸、权利要求6的载体或权利要求8所述的组合物在保证基因编辑过程中的基因组稳定性、提高CAR-T构建过程中T细胞的基因组稳定性或防止改造的CAR-T细胞发生瘤变中的用途。
  17. 制备根据权利要求1-4中任一项所述的融合蛋白的方法，其包括：
    在适合于培养宿主细胞的条件下培养权利要求7所述的宿主细胞；以及
    收获权利要求1-4中任一项所述的融合蛋白。
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