CN116732099A

CN116732099A - Stem cell multiple CRISPR/Cas genome editing method

Info

Publication number: CN116732099A
Application number: CN202310984729.6A
Authority: CN
Inventors: 薛永博; 雷莎莎; 刘康; 时艳
Original assignee: Beisai Hongsheng Beijing Biotechnology Co ltd
Current assignee: Beisai Hongsheng Beijing Biotechnology Co ltd
Priority date: 2023-08-07
Filing date: 2023-08-07
Publication date: 2023-09-12
Anticipated expiration: 2043-08-07
Also published as: CN116732099B

Abstract

The invention relates to the technical field of stem cell gene editing, in particular to a stem cell multiple CRISPR/Cas genome editing method. The stem cell multiplex CRISPR/Cas genome editing method provided by the invention can realize single-step multiplex genome editing in the pluripotent stem cells, has relatively high editing efficiency, greatly simplifies the whole genome editing process, saves the cost and time of gene editing, and reduces repeated passage, culture and editing of the stem cells, thereby reducing the possibility of damaging the integrity of the genome.

Description

Stem cell multiple CRISPR/Cas genome editing method

Technical Field

The invention relates to the technical field of stem cell gene editing, in particular to a stem cell multiple CRISPR/Cas genome editing method.

Background

In recent years, CRISPR/Cas technology has drastically changed the field of gene editing by its simplicity and robustness (Cong, l., ran, f.a., cox, d., lin, s., barretto, r., habib, n., hsu, p.d., wu, x., jiang, w., marraffini, l.a., and Zhang, f. (2013) Multiplex genome engineering using CRISPR/Cas systems, science, 339 (6121), 819-823). However, multiplex genome editing in stem cells, especially pluripotent stem cells (Pluripotent Stem Cells, PSC), has been facing greater challenges due to their inefficiency, particularly with greater difficulty and inefficiency for large fragment knockins. Currently, many researchers have utilized viruses, such as retroviruses, lentiviruses, and adeno-associated viruses (AAV), to increase the efficiency of delivery of genes of interest in PSCs (Han, x., et al (2019). Generation of hypoimmunogenic human pluripotent Stem cells Proceedings of the National Academy of Sciences of the United States of America, 116 (21), 10441-10446; knopp, y., et al (2018). Transient Retrovirus-basal CRISPR/Cas9 All-in-One Particles for Efficient, targeted Gene knockout, molecular therapy, nucleic Acids, 13, 256; martin, r. m., et al (2019), highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 dos-Mediated Homologous recon, cell Stem Cell, 24 (5), 821-828.e5; raguaram, a., et al (2) Therapeutic in vivo delivery of Gene editing agents Cell, 185 (15), 2806-2827. However, this may induce unexpected insertional mutations and tumorigenesis; furthermore, the packaging volume of viruses is quite limited. Also researchers have utilized small molecules to activate the HDR repair pathway or inhibit the NHEJ repair pathway to greatly increase the HDR efficiency in PSCs (Lin, s., et al (2014 b). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 release, ELife, 3, e04766; riesenberg, s., & maric, t. (2018). Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells, nature Communications, 9 (1); zhang, w., et al (2020). A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated gene regulation, ELife, 9, 1-25.). However, the efficacy of these small molecules remains controversial. Therefore, it is of great importance to develop stem cell multiplex CRISPR/Cas genome editing methods that are independent of viruses and small molecule potentiators.

Disclosure of Invention

The invention provides a stem cell multiplex CRISPR/Cas genome editing method.

In order to solve the problem that multiple genome editing across different loci is difficult to realize in stem cells, the invention takes stem cells as a research object, and realizes multiple and synchronous genome editing across different loci in stem cells without using viruses and small molecule synergists by utilizing and optimizing a CRISPR/Cas 9-mediated genome editing method.

Specifically, the invention provides the following technical scheme:

the invention provides a stem cell multiplex CRISPR/Cas genome editing method, which comprises the following steps:

a) Obtaining a Ribonucleoprotein (RNP) complex comprising a CRISPR/Cas endonuclease complexed with a guide RNA (gRNA) that targets a target gene for genome editing;

b) Introducing the ribonucleoprotein complex and a donor template into a stem cell, the donor template comprising a selectable marker gene;

the number m of the types of the selection markers is smaller than the number n of target genes edited by multiple genomes, wherein m is more than or equal to 1 and is an integer, and n is more than or equal to 2 and is an integer.

In the invention, in human-derived pluripotent stem cells (hPCCs), multiple genome editing cannot be realized by using various CRISPR/Cas related plasmids containing Cas and gRNA expression cassettes and optimizing various editing conditions such as the proportion of the plasmids to a donor template, and the implementation of multiple genome editing of stem cells can be remarkably promoted by using CRISPR/Cas RNP (ribonucleoprotein complex) technology. Although CRISPR/Cas RNP technology can improve editing efficiency, positive clones for multiplex genome editing cannot be obtained even with the aid of small molecule potentiators, even if CRISPR/Cas RNP technology is used and nuclear transfection conditions are optimized. The present invention has unexpectedly found that, unlike the case where one selection marker gene is set for each target gene during multiplex genome editing which is generally considered in the art, the number of selection marker genes is appropriately reduced in stem cell multiplex genome editing, i.e., the number of selection markers is controlled to a level less than the target gene factor, which does not affect the editing of target genes for which no selection marker gene is set, but rather is more advantageous in improving the efficiency of multiplex genome editing, and has an important promoting effect on achieving multiplex genome editing of stem cells.

The target gene to which the selection marker gene is not provided is not particularly limited in principle, and any one or more of all the target genes may be used.

In order to simplify gene editing, if multiple genome editing is performed including both gene knockout and knock-in, it is preferable that a selection marker gene is not provided for a target gene for gene knockout, and further, a donor template for the target gene may be omitted.

In the present invention, the number of target genes (i.e., the number of gene editing sites) in multiplex genome editing is several, i.e., several genome edits, and for example, the number of target genes is 3, i.e., three genome edits.

In the present invention, the selectable marker may be any selectable marker that enables positive clone selection including, but not limited to, antibiotic selectable markers (e.g., hygromycin resistance selectable markers, puromycin resistance selectable markers, and the like).

In some embodiments of the invention, m (the number of types of selectable markers) =n (the target base factor for multiplex genome editing) -1.

In some embodiments of the invention, n=3, m=2.

In the present invention, the Cas protein is preferably a Cas9 protein or a protein having the same endonuclease function as the Cas9 protein.

In the stem cell multiplex CRISPR/Cas genome editing method described above, the obtaining of the ribonucleoprotein complex comprises: and respectively mixing and incubating the guide RNA of each target gene with CRISPR/Cas endonuclease to prepare ribonucleoprotein complexes corresponding to the guide RNA, and mixing ribonucleoprotein complexes corresponding to the guide RNA.

Compared with the method for preparing the ribonucleoprotein complex by mixing a plurality of guide RNAs with Cas protein at the same time commonly adopted in the field of multiple genome editing, the method for mixing ribonucleoprotein complexes corresponding to the guide RNAs after each guide RNA is respectively mixed with Cas protein for incubation can be used for further improving the multiple genome editing efficiency of stem cells.

The incubation is preferably performed at room temperature.

In addition, to ensure gene editing efficiency, the prior art has used two or more guide RNAs to target a single target gene. The invention discovers that when the stem cell multiple genome editing method is adopted, one guide RNA is used for targeting one target gene, so that off-target possibly occurring when CRISPR/Cas is used for editing the genome can be obviously reduced, and meanwhile, the multiple editing efficiency is ensured.

Preferably, in the stem cell multiplex CRISPR/Cas genome editing method described above, one guide RNA targeting each target gene is designed for that target gene.

Preferably, the stem cells of the present invention are pluripotent stem cells.

The pluripotent stem cells include Embryonic Stem Cells (ESCs), induced pluripotent stem cells (iPS), or chemically induced pluripotent stem Cells (CiPS).

The invention is not particularly limited as to the animal source of pluripotent stem cells.

In some embodiments of the invention, the pluripotent stem cells are human pluripotent stem cells.

Preferably, in the stem cell multiplex genome editing method described above, the introduction of the ribonucleoprotein complex and the donor template is performed when the stem cells reach 40% -60% confluence (conflux).

The present invention has found that for human pluripotent stem cells (hpscs), the effect of ribonucleoprotein complex and donor template introduction is optimal when they reach 40% -60% confluence, where most hPSC cells are in S/G2 phase, improving the efficiency of HDR.

The method for introducing the ribonucleoprotein complex and the donor template is not particularly limited as long as the ribonucleoprotein complex and the donor template can be transferred into stem cells.

In some embodiments of the invention, ribonucleoprotein complex and donor template introduction employ nuclear transfection techniques. The ribonucleoprotein complex and donor template are mixed and transfected into stem cells using nuclear transfection techniques.

In some embodiments of the invention, the donor template is a plasmid.

The stem cell multiplex CRISPR/Cas genome editing method described above further comprises:

c) Screening positive clones by using a selection marker;

and, d) removing the selection marker gene of the positive clone.

Step c) above allows for a gradual increase in selection pressure during positive clonal selection of the selection marker to allow for recovery and expansion of the desired cells.

Preferably, in step d), the selectable marker gene is removed using the Cre/LoxP recombination system and the HSV-TK/GCV selection system.

After selection of positive clones for gene editing using a selection marker, the selection marker gene in the positive clone is not necessarily present, especially in cell therapy applications, requiring removal of the selection marker gene. The invention uses Cre/loxP recombination system and herpes simplex virus thymidine kinase (HSV-TK)/Ganciclovir (GCV) suicide gene therapy system to remove the selection marker gene introduced in the donor template.

In some embodiments of the invention, the selectable marker gene contained in the donor template is fused to the TK via a T2A/P2A self-cleaving peptide and a cognate loxP site recognized by Cre recombinase is introduced at both ends of the fusion sequence. After transfection of the Cre recombinase nucleus, the flanked fragment will be deleted from the DNA sequence, whereas the cell in principle cannot metabolize the substrate GCV to toxic products due to the loss of TK enzyme, thus enabling selection of positive clones from the cell population from which the selectable marker gene was removed.

Preferably, in the step d), after Cre recombinase nuclear transfection, the transfected product is subjected to GCV screening for 48-72 hours, then cells are collected for subcloning, subcloning is further screened by using GCV, and finally positive clones with the selectable marker genes removed are obtained through identification.

When the Cre/LoxP recombination system and HSV-TK/GCV selection system were used to remove the selectable marker genes, the present invention initially tried a variety of nuclear transfection conditions and GCV selection conditions but none of them could obtain clones that removed two or more selectable marker genes simultaneously. In continuous attempts, the invention surprisingly found that subcloning the Cre recombinase nuclear transfection product subjected to GCV primary screening can significantly improve the removal efficiency of the selectable marker gene, reduce the heterogeneity of clones, and is more beneficial to obtaining clones for removing two or more selectable marker genes simultaneously. By combining the Cre/LoxP recombination system and the HSV-TK/GCV selection system with cell subcloning, the invention realizes the efficient removal of two or more selection marker genes in one step.

Preferably, in step d), after Cre recombinase nuclear transfection, the transfected product is cultured in a medium containing GCV for 48-72h, and the cells are collected for subcloning.

Further preferably, in step d), 48-72 hours after electroporation of Cre recombinase nuclear transfection, GCV is added for 48-72 hours and the cells are harvested for subcloning.

Preferably, the concentration of the GCV screen is 0.2-1.0. Mu.M.

The above-described identification may be performed by methods commonly used in the art, such as PCR identification, DNA sequencing, and the like.

The invention further provides a method for preparing a low-immunogenicity pluripotent stem cell, comprising: the stem cell multiplex CRISPR/Cas genome editing method is adopted to carry out genome editing on the pluripotent stem cells, so that the immunogenicity of the pluripotent stem cells is reduced.

The stem cell multiplex CRISPR/Cas genome editing method is adopted to carry out genome editing on the pluripotent stem cells, so that not only can the efficiency of the multiplex genome editing be improved, but also the selection marker genes can be removed before the GMP-class functional cells are produced by the stem cells subjected to the gene editing. The method can be used for preparing the low-immunogenicity universal pluripotent stem cell line, has the advantages of high editing efficiency, lower difficulty and simpler operation, and is an ideal method for preparing the low-immunogenicity pluripotent stem cell line in a mode meeting or exceeding GMP requirements. In addition, the low immunogenicity of the universal pluripotent stem cell lines can better overcome the problem of immune rejection in allograft, in the field of regenerative and transformation medicine, providing safe, stable and lower cost stem cell products.

In the above method, the reduction of the immunogenicity of the pluripotent stem cells can be achieved by genome editing knock-in of genes that can reduce the immunogenicity of the pluripotent stem cells and/or knock-out of genes that prevent the reduction of the immunogenicity of the pluripotent stem cells. The gene that reduces the immunogenicity of the pluripotent stem cells and the gene that prevents the reduced immunogenicity of the pluripotent stem cells may be any gene known in the art to have the above-described functions, or may be a newly discovered gene having the above-described functions.

In some embodiments of the invention, the genome editing comprises: the fusion protein B2M-HLA E gene is knocked in, the CD47 gene is knocked in, and the CIITA gene is knocked out.

The fusion protein B2M-HLA E is a fusion protein of B2M and HLA-E. Preferably, an HLA-G signal peptide is linked to the N-terminus of B2M-HLA E.

By utilizing the stem cell multiplex CRISPR/Cas genome editing method, the invention realizes triple genome editing of fusion protein B2M-HLA E gene knock-in, CD47 gene knock-in and CIITA gene knock-out synchronously in the pluripotent stem cells, and greatly improves the efficiency of stem cell gene editing.

In the above method for preparing a low-immunogenicity pluripotent stem cell, the donor template comprises a first donor template and a second donor template;

The first donor template comprises a B2M-HLA E expression cassette and a first selectable marker gene;

the second donor template includes a CD47 expression cassette and a second selectable marker gene.

According to the invention, when triple genome editing of fusion protein B2M-HLA E expression cassette knocking-in, CD47 expression cassette knocking-in and CIITA gene knocking-out is carried out, the CIITA gene knocking-out is not screened by using a selection marker gene, a donor template for CIITA gene knocking-out is omitted, the efficiency of triple genome editing can be obviously improved by using the first donor template and the second donor template, and triple genome editing of fusion protein B2M-HLA E expression cassette knocking-in, CD47 expression cassette knocking-in and CIITA gene knocking-out can be realized in one-step transfection.

In some embodiments of the invention, the genomic locus knocked in by the fusion protein B2M-HLA E expression cassette is the B2M gene locus. The genomic site knocked in by the CD47 expression cassette is the AAVS1 site.

In some embodiments of the invention, the nucleotide sequence of the sgRNA targeting the B2M gene locus is shown in SEQ ID NO. 1. The nucleotide sequence of sgRNA targeting AAVS1 site is shown as SEQ ID NO. 2. The nucleotide sequence of the sgRNA of the targeting CIITA gene is shown as SEQ ID NO. 3.

In some embodiments of the invention, the fusion protein B2M-HLA E expression cassette comprises an HLA-G signal peptide gene, a B2M mature protein gene, a linker sequence, and an HLA-E gene, a polyA sequence.

In some embodiments of the invention, the CD47 expression cassette comprises a CAG promoter, a CD47 gene and a polyA sequence.

Preferably, the donor template further comprises an upstream and downstream homology arm to the knock-in genomic locus.

In some embodiments of the invention, the first selectable marker gene is driven by an EF1a promoter. The second selectable marker gene is driven by an endogenous AAVS1 promoter.

In some embodiments of the invention, the first selectable marker and the second selectable marker are two different selectable markers, for example, two different antibiotic resistance selectable markers.

The present invention induces two longer biallelic knockins and one biallelic knockout with relatively high efficiency by single step nuclear transfection (one single step nucleofection) of pluripotent stem cells, thereby obtaining low immunogenicity pluripotent stem cells that can be used as a feedstock for cell therapy. In the above method, the triple genome editing efficiency can reach 18.2% after the whole resistance screening period and PCR identification (4 expected clones are obtained from 22 clones and carry homozygous HLA-E and CAG-CD47 knockins and complete CIITA knockouts).

The invention also provides a low-immunogenicity pluripotent stem cell, wherein CIITA genes and B2M genes of the low-immunogenicity pluripotent stem cell are inactivated and carry a homozygous HLA-E expression cassette and a homozygous CD47 expression cassette.

Preferably, the fusion protein B2M-HLA E expression cassette comprises an HLA-G signal peptide gene, a B2M mature protein gene, a linker sequence and an HLA-E gene.

Preferably, the CD47 expression cassette comprises a CAG promoter and a CD47 gene.

Preferably, the genomic locus knocked-in by the HLA-E expression cassette is a B2M gene locus, endogenous B2M expression is disrupted, and the knocked-in HLA-E gene drives expression under the endogenous B2M promoter. The genomic site knocked in by the CD47 expression cassette is the AAVS1 site.

Preferably, the low-immunogenicity pluripotent stem cells are prepared by the preparation method of the low-immunogenicity pluripotent stem cells.

The beneficial effects of the invention at least comprise: the stem cell multiplex CRISPR/Cas genome editing method provided by the invention can realize single-step multiplex genome editing in stem cells, has relatively high editing efficiency, does not need to use viruses or micromolecular synergists, not only greatly simplifies the whole genome editing process, thereby saving the cost and time of gene editing, but also reduces repeated passage, culture and editing of the stem cells, and thus reduces the possibility of damaging the integrity of the genome.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a B2M site knock-in B2M-HLA E fusion protein expression cassette according to example 1 of the present invention.

FIG. 2 is a schematic representation of CIITA gene knockout in example 1 of the present invention.

FIG. 3 is a schematic representation of the AAVS1 site knock-in CD47 expression cassette of example 1 of the present invention.

FIG. 4 shows the morphology of positive clones obtained by antibiotic selection in example 1 of the present invention.

FIG. 5 shows the genotyping results of the positive clones obtained by antibiotic selection in example 1 of the present invention, wherein the clone that was successfully knocked in the B2M-HLA E fusion protein expression cassette was amplified to obtain a 1.6kb fragment, whereas the fragment that was not knocked in was amplified to obtain a 2.1kb fragment; cloning amplification with successful knock-in of the CD47 expression cassette gives 1.3kb, and amplification without knock-in gives 1.9kb; wherein, clone 9, 21, 10, 14, 33 is a bi-allele B2M-HLA E fusion protein expression cassette knock-in and a CD47 expression cassette knock-in.

FIG. 6 shows the sequencing results of clones that were successfully knocked into the B2M-HLA E fusion protein expression cassette and clones that were successfully knocked into the CD47 expression cassette in example 1 of the present invention.

FIG. 7 shows the sequencing results of CIITA knockout clone in example 1 of the present invention.

FIG. 8 is a schematic of a strategy for removing antibiotic selectable marker genes using the Cre/LoxP recombination system and HSV-TK/GCV selection system in example 1 of the present invention.

FIG. 9 shows the results of PCR identification of antibiotic selectable marker genes with the B2M site and AAVS1 site removed using method 1 (Cre nuclear transfection and GCV screening) in example 1 of the present invention, showing that all clones did not remove antibiotic selectable marker genes with the B2M site and AAVS1 site.

FIG. 10 shows the results of PCR identification of antibiotic selection marker genes removed at the B2M site and AAVS1 site using method 2 (subcloning) in example 1 of the present invention, showing that clone #25 and #32 still did not remove antibiotic selection marker genes, and that the remaining clones successfully removed antibiotic selection marker genes.

FIG. 11 shows the sequencing results of the antibiotic selectable marker gene with the AAVS1 site removed using method 2 (subcloning) in example 1 of the present invention.

FIG. 12 shows the sequencing result of the antibiotic selectable marker gene removed at the B2M site using method 2 (subcloning) in example 1 of the present invention.

FIG. 13 shows the results of a karyotype analysis of positive clones obtained in example 1 of the present invention with the antibiotic selectable marker gene removed.

FIG. 14 shows the low immunogenicity of hESC (B2M) after IFN-gamma treatment in example 2 of the invention ^Etrimer CIITA ^null CD47 ^OE The flow assays for HLA-A, B, C expression (a) and CD47 expression (B) of hescs, i.e., U-hescs) and wild-type (WT) hescs, are indicated on the right side of each figure relative to the Mean Fluorescence Intensity (MFIs) of wild-type hescs.

FIG. 15 shows the low immunogenicity hESC (B2M) of example 2 of the present invention ^Etrimer CIITA ^null CD47 ^OE hESC,) and wild-type (WT) hESC differentiation to CD14 ⁺ Morphology detection of monocytes, wherein bright field imaging is 0-14 day cell morphology, scale 100 μm.

FIGS. 16 and 17 are graphs showing detection of hypoimmunogenic hESC (B2M) using CD14 expression in example 2 of the present invention ^Etrimer CIITA ^null CD47 ^OE hESC) and wild-type (WT) hESC differentiation to CD14 ⁺ Efficiency of monocytes.

FIG. 18 shows the low immunogenicity hESC (B2M) of example 2 of the present invention ^Etrimer CIITA ^null CD47 ^OE hESC) and wild-type (WT) hESC differentiation to CD14 ⁺ After monocytes, HLA-DR cell surface protein expression was examined by FCM.

FIG. 19 shows the use of CFSE in wild-type hESC and hypoimmunogenic hESC (B2M) in example 3 of the invention ^Etrimer CIITA ^null CD47 ^OE hESC) detection of proliferative CD8 on differentiated DA progenitor cells ⁺ T cell response to HLAs.

FIG. 20 shows the use of CFSE in wild-type hESC and hypoimmunogenic hESC (B2M) in example 3 of the invention ^Etrimer CIITA ^null CD47 ^OE hESC) for detecting proliferative CD8 ⁺ T cell response to HLAs.

FIG. 21 shows the use of CFSE in wild-type hESC and hypoimmunogenic hESC (B2M) in example 3 of the invention ^Etrimer CIITA ^null CD47 ^OE hESC) detection of proliferative CD4 on differentiated monocytes ⁺ T cell response to HLAs.

The T cells cultured alone in fig. 19, 20, 21 served as negative controls; CD3/CD28 treated T cells served as positive control.

FIG. 22 shows the medium and low immunogenicity of hESC (B2M) in example 3 of the invention ^Etrimer CIITA ^null CD47 ^OE hESC) were incubated together, NK cells were assayed for cytotoxicity.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the following examples, cas9/gRNA complex (ribonucleoprotein complex) was transfected into cells by using a nuclear transfection system (Lonza) 2B nuclear transfecter, procedure B016) according to the manufacturer's instructions. After Cas9 is incubated with gRNA to obtain Cas9/gRNA complex, the donor plasmid can be directly added to the nuclear transfection buffer and then mixed with Cas9/gRNA complex to obtain a nuclear transfection mixture.

EXAMPLE 1 multiple genome editing of Stem cells

1. Construction of donor templates

1. Preparation of first donor template (B2M-HLA E fusion (Etrimer) vector): construction of vectors is described in the literature. Amplifying the left and right arms of B2M from genomic DNA of the human embryonic stem cell line H1; amplification of HLA-E from hESC (H1) cDNA; B2M signal peptide- -HLA-G signal peptide- -linker sequence- -B2M mature protein- -linker sequence was synthesized by the company (GENERAL biology); amplifying EF1a promoter from Addgene 60955; hygromycin resistance gene (HygroR) was synthesized by company (GENERAL biology); HSV-TK sequences were amplified from Addgene 165081. The two sides of HygroR-HSV-TK driven by EF1a promoter are provided with the same-directional loxP site.

Each of the above fragments was cloned into pUC57 vector backbone using In-fusion (Takara) and performing seamless cloning according to the manufacturer's instructions.

2. Preparation of the second donor template (CAG-CD 47 vector): amplifying AAVS1 left and right arms from H1 genomic DNA; amplifying puromycin resistance gene (PuroR) from adedge 60955; digesting Addgene 27077 to obtain a CAG promoter; the CD47 gene was amplified from hESC (H1) cDNA. HSV-TK sequences were amplified from Addgene 165081. The PuroR-HSV-TK is flanked by orthologous loxP sites.

2. Preparation of Cas9-gRNA complexes and Nuclear transfection

1. Hescs were cultured in mTesR1 (Stem Cell technology) until about 50% confluency was reached prior to nuclear transfection.

2. Each mixture of gRNA and Cas9 is prepared separately, i.e., B2M gRNA, CIITA gRNA, AAVS1 gRNA are mixed and incubated with Cas9, respectively, to prepare complexes of each gRNA and Cas 9. The specific method of mixed incubation is as follows: 15 pmol of Cas9 protein (Thermo, A36496) was mixed with 90 pmol of synthetic gRNA (Genesril) in a sterile tube of 1.5. 1.5 mL and nuclear transfection buffer was added to 10. Mu.L. The reaction mixture was incubated at room temperature for about 30 minutes. In another 1.5 mL sterile tube, 2 μg of donor plasmid was added to the remaining nuclear transfection buffer.

The sequences of the grnas used were as follows:

B2M gRNA(SEQ ID NO.1)：GAGTAGCGCGAGCACAGCTA；

CIITA gRNA(SEQ ID NO.3)：GATATTGGCATAAGCCTCCC；

AAVS1 gRNA(SEQ ID NO.2)：GTCACCAATCCTGTCCCTAG。

the target site cleavage efficiency of each gRNA was predicted to be: B2M gRNA,48.12%; CIITA gRNA,49.94%; AAVS1 gRNA,61.02%.

3. Hescs (1 well in 6 well plate) were digested with 1ml Accutase for 3 min, then the digestion reaction was stopped with 1ml mTesR1. Using 1/3 of the cells (about 1X 10) ⁶ ) Nuclear transfection was performed.

4. Cas9-gRNA complex and donor plasmid were transfected into stem cells using a 2B nuclear transfecter (Lonza, B016 program) following the manufacturer's instructions.

5. Immediately after nuclear transfection, cells were transferred to pre-warmed medium (10. Mu.M ROCK inhibitor Y27632 or chemical cocktail CET (50 nM Chroman 1, 5. Mu.M Emricasan and 0.7. Mu.M Trans-ISRIB) was added to the medium) to promote cell survival after nuclear transfection.

6. Antibiotics were added 48-72 hours after electroporation to remove unedited cells.

During the selection process, the selection pressure is gradually increased. The donor plasmid HLA-E fusion vector carries the hygromycin resistance gene (driven by the EF1a promoter) and the donor plasmid CAG-CD47 carries the puromycin resistance gene (controlled by the endogenous AAVS1 promoter). In the antibiotic selection, 10. Mu.g/mL hygromycin was used for 2 days, followed by 10. Mu.g/mL hygromycin+0.2. Mu.g/mL puromycin for 2 days, followed by 20. Mu.g/mL hygromycin+0.5. Mu.g/mL puromycin for 4 days, and finally 40. Mu.g/mL hygromycin+1. Mu.g/mL puromycin was used until single clones were observed.

7. Single clones were selected and incubated in 40. Mu.g/mL hygromycin+1. Mu.g/mL puromycin.

8. PCR identification and Sanger sequencing were performed for B2M, AAVS site knock-in and CIITA site knock-out.

3. Antibiotic resistance gene removal

Although different antibiotic resistance genes were introduced at the B2M and AAVS1 sites, they had LoxP elements on both sides in the same direction. Thus, in order to shorten the editing process, the present invention seeks to remove both antibiotic resistance genes simultaneously rather than stepwise. However, during the trial it was found that after several rounds of repeated experiments, the GCV treatment did not ensure that the cells completely lost TK expression, and that the TK fragment was still amplified from the clone with relatively low intensity after Cre treatment. To reduce the heterogeneity of these clones and to obtain the desired clones, they were further subcloned.

The Cre recombinase nuclear transfection, GCV treatment and subcloning were performed sequentially to remove the antibiotic resistance gene as follows:

1. the B2M, AAVS1 site knock-in and CIITA site knock-out hypoimmunogenic hESCs obtained in step two above were cultured in mTesR1 (Stem Cell technology) until they reached about 70% confluency.

2. Hescs (1 well in 6 well plate) were digested with 1 mL Accutase for 3 min and then quenched with 1 mL mTesR1. Nuclear transfection was performed using 1/3 of the cells.

3. 40 pmol Cre protein (Sigma-Aldrich, SCR 508) was mixed with 100. Mu.L of nuclear transfection buffer in a 1.5 mL sterile tube.

4. Nuclear transfection was performed using a 2B nuclear transfecter (Longza, inc. (Lonza), procedure B016) according to the manufacturer's instructions.

6. Unedited cells were removed by adding 0.5. Mu.M GCV 48-72 hours after electroporation and culturing for 72 hours.

7. Cells were digested and harvested and plated into 96-well plates for subcloning at a density of 0.5-1 cells/well.

Note that: 10. Mu.M ROCK inhibitor Y27632 or CET (50 nM Chroman 1, 5. Mu.M Emricasan and 0.7. Mu.M Trans-ISRIB) was added to the medium to promote cell survival after nuclear transfection.

8. After 72 hours the medium was changed and 0.5. Mu.M GCV was supplemented.

9. The medium was changed every 24 hours and 0.5. Mu.M GCV was supplemented until the clones grew.

10. PCR identification and Sanger sequencing were performed for removal of the antibiotic resistance gene at the B2M, AAVS site.

The reaction conditions for PCR identification were as follows:

1. B2M site PCR:

for the absence of insertion, the products obtained for B2M-nest-F1 and B2M-Cy-R were 2112bp.

For the presence of HLA-E expression cassette insertion, B2M-nest-F1 and HLA-E-up-R gave a product of 1654bp.

B2M-nest-F1(SEQ ID NO.4)：GATGCAGTCCAAACTCTCAC；

B2M-Cy-R(SEQ ID NO.5)：GCTCTGGAGAATCTCACGCAG；

HLA E-up-R(SEQ ID NO.6)：CACGGAAGTGTGGAAATACTTC。

The PCR reaction system and the reaction procedure are shown in Table 1.

TABLE 1

2. CIITA site PCR:

the primer CIITA-Lu-F and CIITA-Cy-R were used to obtain 1904bp fragments for sequencing verification.

CIITA-seq-1(SEQ ID NO.7)：GCAGGACTTCAGTCAGACTGTC；

CIITA-Cy-R(SEQ ID NO.8)：GCACTTTGCTAGCCAAGCAG。

The PCR reaction system and the reaction procedure are shown in Table 2.

TABLE 2

3. AAVS1 site PCR:

for the case where no insertion exists, AAVS1-nest-F2 and AAVS1-nest-R2 result in 1890bp;

for the case where an insertion was present, CD47-Down-F and AAVS1-nest-R2 gave 1218bp;

AAVS1-nest-F2(SEQ ID NO.9)：

CTTTGAGGCTCTTACTGGCTTCTG；

AAVS1-nest-R2(SEQ ID NO.10)：GATGAGTTTGCCAACAGTC；

CD47-down-F(SEQ ID NO.11)：GAGTATCTTAGCTCTAGCAC。

the PCR reaction system and the reaction procedure are shown in Table 3.

TABLE 3 Table 3

4. Removal of EF1a-hygroR (hygromycin resistance gene) -T2A-TK at the B2M site

For the case of removal failure, TK-PCR-F3 and B2M-confirm-R gave 1044bp bands, and no band was found upon successful removal.

TK-PCR-F3(SEQ ID NO.12)：GCATGTCTTTATCCTGGATTAC；

B2M-confirm-R(SEQ ID NO.13)：

GAAGTCCACACAGCTTCCAGTC。

The PCR reaction system and the reaction procedure are shown in Table 4.

TABLE 4 Table 4

5. T2A-puroR (puromycin resistance gene) -P2A-TK removal at AAVS1 site

For the case of removal failure, AAVS1-seq-1 and puro-M-R gave a 489bp band, and no band was found upon successful removal.

AAV S1-seq-1(SEQ ID NO.14)：GAGAGCCTAGCTAGTCTTCTTC；

puro-M-R(SEQ ID NO.15)：GTGAGGAAGAGGTTCTTGCAGC。

The PCR reaction system and the reaction procedure are shown in Table 5.

TABLE 5

The main experimental results of the stem cell multiplex genome editing of the present invention are summarized as follows:

in the Ethimer construct, the HLA-G signal peptide is fused to structural element B2M, and subsequently to the HLA-E genomic sequence, and the HLA-E protein plays a key role in inhibiting NK cell-dependent lysis and presenting the HLA-G peptide. The Etrimer donor template was integrated into B2M exon 1 by CRISPR/Cas9 mediated editing (fig. 1). This insertion has a dual role, one is to disrupt endogenous B2M expression and the other is to allow HLA-E expression under the native B2M promoter. In CIITA knockout, CIITA knockout was achieved using gRNA targeting exon 3 in the absence of donor template (fig. 2). In the CD47 overexpression construct, expression of CD47 was controlled by the constitutive strong promoter CAG (fig. 3). The CD47 overexpression construct was intended to integrate into the AAVS1 safe harbor site (safe harbor) (fig. 3). In addition, unlike the Etrimer design, where expression of the selectable marker gene is driven by the EF1a promoter, the selectable marker gene in the CD47 construct is driven by the endogenous AAVS1 promoter after integration.

The present invention has attempted to use CRISPR/Cas 9-related plasmids (e.g., PX339 in adedge) comprising Cas9 (D10A) mononickase and a gRNA expression cassette in the early stages of development, but none have achieved any biallelic insertion of an Etrimer at the B2M site, even one insertion. Although attempts were made to optimize the ratio between Cas9 vector and donor, methods using plasmids for gene editing were still quite inefficient in stem cells. Similar situation is also observed when Cas9 wild-type vector (such as PX459 in adedge) is used instead. Thus, the present invention contemplates the use of Cas9/gRNA RNP (RNP method) instead of CRISPR/Cas9 plasmid and it was observed that RNP method is significantly superior to plasmid method in obtaining double allele knock-in or knock-out in stem cells.

Based on the introduction of the RNP method, the present invention attempts to simultaneously make triple gene modification in stem cells. Initially, donor templates carrying selectable marker genes were designed to enrich for CIITA knockout clones, but when applied simultaneously with the other two donor templates at B2M and AAVS1 sites, multiple attempts after the selection process still failed to obtain any viable clones. Considering that HDR is more effective in S/G2 phase, cell density of cell nucleus transfection is further regulated, and small molecule synergists such as identified RS-1 and Nocodazole (Nocodazole) which can promote HDR are supplemented in cell culture before and after cell nucleus transfection, only few clones are formed after drug screening, but the final results are all false positive clones.

At this time, it seems that the triple gene modification cannot be achieved. However, the present invention accidentally attempted to remove CIITA knockout donor templates from nuclear transfection mixtures, and unexpectedly found that genetic modifications spanning these three sites could occur simultaneously, so that CIITA knockout clones could be selected by selection markers for B2M and AAVS 1. In fact, a large number of clones with representative morphological features were formed after the selection process using the strategy described above (FIG. 4).

Cloning was further performed: 1) PCR analysis was performed using primers complementary to DNA sequences located outside the homology arm framework to exclude non-specific integration (fig. 5); 2) Sanger sequencing to confirm the sequence of the desired insert, knockout (FIGS. 6, 7); 3) Karyotyping to detect genome integrity; 4) Whole Genome Sequencing (WGS) to assess the potential impact of gene editing. The results of gene editing and the gene editing efficiency of the finally obtained 4 positive clones are summarized in Table 6. Overall, the efficiency of triple gene editing (18.2%) was significantly more efficient after removal of CIITA knockdown donor templates than previous designs using three donor templates (0%), and these clones maintained normal karyotype.

TABLE 6

Next, the selection marker genes at the B2M and AAVS1 sites were removed using Cre recombinase and prodrug GCV (fig. 8). The amount of Cre recombinase required at nuclear transfection is less than that required for incubation (greater amount of Cre recombinase), and can more effectively mediate deletion of the DNA sequence flanked by LoxP. Following Cre recombinase nuclear transfection, the cells are treated with GCV to select cells that no longer carry TK DNA sequences. Since 1. Mu.M GCV was suitable for selection at the B2M site and 10. Mu.M GCV was suitable for selection at the AAVS1 site after Cre recombinase nuclear transfection, the GCV concentration was difficult to equilibrate. Finally, 0.2. Mu.M-1. Mu.M GCV was used for selection, considering that higher concentrations of GCV would be toxic to these clones. However, the combination of 40 pmol Cre recombinase nuclear transfection and 0.5. Mu.M GCV selection was able to remove the resistance markers at a single site, but not both sites at the same time (FIG. 9). Subsequently, nuclear transfection was performed using three and five times Cre recombinase, with the aim of increasing recombination efficiency, but similar results to 40 pmol Cre recombinase nuclear transfection were observed (FIG. 9). The present inventors have surprisingly found that the TK fragments of these clones can still be amplified at relatively low intensities after treatment with Cre recombinase and have accordingly attempted subcloning to reduce the heterogeneity of these clones. The results demonstrate that subclones help select the desired clone, and several positive clones were determined after PCR analysis and Sanger sequencing (fig. 10, 11, 12). The positive clones were subjected to a karyotyping analysis to examine the genome integrity, and the results are shown in FIG. 13.

The finally obtained hypoimmunogenic hESC cells of the invention carry a homozygous Ethimer cassette (5.2 kb) at the B2M site, a homozygous CD47 overexpression cassette (overexpression cassette) (5.8 kb) at the AAVS1 site, and have a homozygous CIITA knockout, designated B2M ^Etrimer CIITA ^null CD47 ^OE hescs, U-hescs for short.

Example 2 verification of expression of HLA class I/II molecules and immunomodulatory factor CD47 in engineered Low immunogenicity pluripotent Stem cells

The expression verification of HLA class I/II molecules and immunomodulatory factor CD47 was performed on the low-immunogenicity hescs obtained by multiplex genome editing in example 1 using flow cytometry analysis methods, specifically as follows:

the hypoimmunogenic hescs were dissociated with accutase for 3 min at 37 ℃ and the single cells were washed 1 time with Phosphate Buffer (PBS). The hypoimmunogenic hESC suspension was incubated with FACS antibodies (1:20 dilution) in Phosphate Buffer (PBS) containing 1% fetal bovine serum for 20 minutes at room temperature and washed twice before analysis on BD FACSLyric (BD Biosciences). Data was drawn using FlowJo software (BD). For the IFN-gamma treatment group, low immunogenicity hESC (B2M) was treated with 300 ng/mL IFN-gamma (peprotech Co., pai Pratech) ^Etrimer CIITA ^null CD47 ^OE hESC) and wild-type (WT) hESC for 48 hours each, however Cells were then harvested for flow cytometry analysis. The antibodies used were: anti-HLA-A, B, C antibodies conjugated to APC (biological), clone W6/32), FITC anti-human HLA-DR antibody (biological), LN3, perCP/cyanne 5.5 anti-human HLA-E antibody clone (biological), 3D12, PE anti-human CD47 antibody (biological), clone CC2C6, APC anti-human CD14 antibody (biological), clone 63D3, PE anti-human CD8 antibody (biological), clone SK1, APC anti-human CD4 monoclonal antibody (eBioscience ™, clone OKT 4) and their corresponding isotype controls (biological).

Typical HLA class I molecules are highly reactive to cytokine stimulation and their expression is strongly up-regulated by Interferon (IFN) - γ stimulation at both mRNA and protein levels. To increase HLA cell surface expression, cytokine IFN-gamma treatment concentrations were measured and 300 ng/ml IFN-gamma treatment was used to up-regulate HLA class I molecule expression levels. The results showed that even under IFN-. Gamma.treatment, in B2M ^Etrimer CIITA ^null CD47 ^OE No surface-typical HLA class I molecules were detected in hescs (fig. 14), confirming that these cell lines have completely knocked out B2M.

HLA class II expression was eliminated by targeting CIITA in hescs. CIITA (major histocompatibility complex HLA class II transactivator) genes are known to be critical for transcription of HLA class II genes and to be in HLA class II ⁺ Specific expression in cells and tissues. The loss of function of the CIITA gene results in a lack of HLA class II due to a lack of antigen presentation capacity through HLA class II. To demonstrate the loss of HLA class II expression, low-immunogenicity hescs and wild-type hescs were differentiated to CD14 using a stem diff monocyte kit (stem cell technologies (STEMCELL Technologies)) according to the manufacturer's protocol ⁺ Monocytes, known as CD14 ⁺ Monocytes express HLA class I and class II genes simultaneously. After differentiation for 14 days or more (FIG. 15), CD14 was confirmed by FCM using an anti-CD 14 antibody ⁺ Percentage of monocytes (fig. 16, 17). These results indicate that gene editing does not affect monocyte differentiation. Then, check by FCMThe expression of HLA-DR cell surface proteins was measured, and cell surface expression of HLA-DR proteins was lost in low immunogenicity hESC-derived monocytes, but not in wild-type monocytes (FIG. 18).

The overexpression of CD47 was further confirmed by Flow Cytometry (FCM) (fig. 14), which indicated that CD47 levels of hypo-immunogenic hESC expression were significantly elevated, with CD47 targeted for integration into AAVS1 safe harbor sites.

Example 3B 2M ^Etrimer CIITA ^null CD47 ^OE In vitro identification of immunogenicity of hESC and differentiated DA progenitor cells thereof

For B2M ^Etrimer CIITA ^null CD47 ^OE hescs and their ability to evade adaptation of differentiated DA progenitor cells and innate immune responses were tested in vitro as follows:

1. in vitro immunoreactivity under NK cells

NK92 cells were used as effector cells, low immunogenicity and wild-type hescs as target cells. Low immunogenicity and wild-type hescs treated with 300 ng/mL IFN- γ for 48 hours were target cells; will be 3X 10 ⁴ Target cells and NK92 cells of individual cells/well were co-incubated in 200 μl NK92 cell medium in 96-well plates for 20 hours at the indicated effector cells/target cell ratio, and then supernatants were harvested; after co-incubation, supernatants were collected and analyzed by LDH cytotoxicity detection kit (Beyontime) according to the manufacturer's instructions. NK92 cell medium (RPMI-1640) was used as background control. NK92 cells cultured alone or target cells cultured alone were used as controls for spontaneous LDH release. Target cells lysed at the endpoint were used as maximum LDH release; the samples were on OD at Thermo Scientific ™ Varioskan ™ LUX ₄₉₀ Analysis at nm. Data were normalized and analyzed using Graphpad prism software.

2. In vitro immunoreactivity under T cells

hESC, hESC-derived monocytes, hESC-derived DA progenitor cells were used as target cells. 10 ten thousand target cells were placed on a 24-well plate and treated with IFN-gamma (300 ng/ml) for 48 hours prior to detection. On day 0 of co-incubation, isolated CD4+/CD8+ T cells were labeled with CellTraceTM CFSE (Semer Feishmania technologies Co., ltd. (ThermoFisher Scientific)) according to the manufacturer's instructions. Adherent hescs and hESC-derived DA progenitors were treated with mitomycin C and washed 3 times with PBS, and then co-incubated with 500k CFSE-labeled T cells in T cell medium supplemented with 20U/ml IL-2 for 5 days. T cells were then stained with anti-CD 4/CD8 antibodies and CFSE intensities were analyzed on BD FACSLyric (BD Biosciences). T cells cultured for 5 days in the absence of target cells were used as negative controls. T cells treated with Dynabeads ™ human T-Activator CD3/CD28 beads (DynabeadsTM Human T-Activator CD3/CD28 beads) (Sesameimer Feier technologies (ThermoFisher Scientific)) for 5 days were used as positive controls. Data was drawn using FlowJo software (BD).

Knockout of B2M in hescs is expected to eliminate surface expression of HLA class I molecules and avoid CD8 ⁺ Direct killing activity of T cells. To investigate whether removal of polymorphic HLA molecules was sufficient to prevent T cell mediated immune responses, low immunogenicity and wild-type hescs were combined with allogeneic CD8 from healthy donors ⁺ T cells were co-cultured. In vitro T cell immunoassays were further performed: t cell proliferation. It has been reported that undifferentiated hescs have limited immunogenicity, and thus, hescs are differentiated into DA progenitor cell derivatives expressing only HLA class I prior to determining their immunoreactivity. Pretreatment of low immunogenicity and wild-type hESC differentiated DA progenitor cells with IFNgamma followed by labeling with carboxyfluorescein succinimidyl ester (CFSE) allogeneic CD8 ⁺ T cells were co-cultured for 5 days. The CFSE signal dilution of T cells was then analyzed by flow cytometry as CD8 ⁺ Readings of T cell proliferation in T cell subpopulations.

The results show (FIG. 19), total proliferation CD8 when incubated with low immunogenicity of hESC-differentiated DA progenitor cells compared to wild-type hESC-differentiated DA progenitor cells (13.7%) ⁺ The percentage of T cells was reduced (1.67%).

Since DA progenitor cell induction is time consuming, the present invention directly performs T cell immunoassays on undifferentiated hescs. At and CD8 ⁺ hESC was subjected to prior to T cell co-culture Mitomycin C treatment to inhibit proliferation. The results show (FIG. 20), CD8 at low immunogenicity hESC compared to wild-type hESC (9.7%) ⁺ Proliferation of T cells was significantly lower (0.7%). This trend is associated with CD8 on DA progenitor cells ⁺ The results of the T cell proliferation assay were consistent.

To evaluate the immunogenic effect of CIITA knockout on hescs, low-immunogenicity hescs and wild-type hescs were differentiated to HLA class I and class II expressing CD14 following ifnγ stimulation as described above ⁺ Monocytes. Since HLA class II binding is specific for CD4 but not for CD8, allogeneic CD4 from healthy donors is used ⁺ T cells were subjected to CSFE analysis. CD4 ⁺ Immunoassay for T cell proliferation follows CD8 ⁺ T cells are similar in pattern. In CD4 ⁺ The same trend was also observed in T cells. As expected, see FIG. 21, HLA class II expressing WT cells stimulated CD4 ⁺ Proliferation of helper T cells (45%) while CIITA-deficient hypoimmunogenic hescs were significantly reduced (21%). The results indicate that depletion of HLA class II molecules by CIITA KO method is effective in inhibiting CD4 in addition to HLA class I depletion ⁺ T cell response.

NK cell-mediated killing plays an important role in immune rejection. In addition, donor cells without HLA class I surface expression can trigger NK cell mediated immune attacks through a lost self response. To evaluate the response of NK cells to hypoimmunogenic hESCs, NK92 cells were compared with WT, B2M ^null 、B2M ^Etrimer CIITA ^null CD47 ^OE hescs were incubated together. After co-incubation with NK92 cells, apoptotic hESC cells were tested for release of Lactate Dehydrogenase (LDH) to quantify NK cell cytotoxicity. The result shows that when the catalyst is combined with B2M ^null 、B2M ^Etrimer CIITA ^null CD47 ^OE When hescs were co-incubated, NK cell cytotoxicity was reduced (fig. 22).

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A stem cell multiplex CRISPR/Cas genome editing method, comprising:

a) Obtaining a ribonucleoprotein complex comprising a CRISPR/Cas endonuclease complexed with a guide RNA that targets a target gene for genome editing;

2. The stem cell multiplex CRISPR/Cas genome editing method according to claim 1, wherein the obtaining of the ribonucleoprotein complex comprises: and respectively mixing and incubating the guide RNA of each target gene with CRISPR/Cas endonuclease to prepare ribonucleoprotein complexes corresponding to the guide RNA, and mixing ribonucleoprotein complexes corresponding to the guide RNA.

3. The stem cell multiplex CRISPR/Cas genome editing method according to claim 1, wherein the stem cell is a pluripotent stem cell;

and/or, the introduction of the ribonucleoprotein complex and the donor template is performed when the stem cells reach 40% -60% confluence.

4. The stem cell multiplex CRISPR/Cas genome editing method according to any one of claims 1 to 3, further comprising:

c) Screening positive clones by using a selection marker;

and, d) removing the selection marker gene of the positive clone.

5. The stem cell multiplex CRISPR/Cas genome editing method according to claim 4, wherein in step d), the selection marker gene is removed using Cre/LoxP recombination system and HSV-TK/GCV selection system.

6. The stem cell multiplex CRISPR/Cas genome editing method according to claim 5, wherein in step d), after Cre recombinase nuclear transfection, the transfected product is first GCV screened for 48-72 hours, then cells are collected for subcloning, subcloning is further screened using GCV, and finally positive clones with the selection marker gene removed are identified.

7. A method of preparing a low immunogenicity pluripotent stem cell, comprising: the stem cell multiplex CRISPR/Cas genome editing method of any one of claims 1 to 6 is used for genome editing of pluripotent stem cells, so that immunogenicity of the pluripotent stem cells is reduced.

8. The method of preparing a low immunogenicity pluripotent stem cell according to claim 7, wherein the genome editing comprises: the fusion protein B2M-HLA E gene is knocked in, the CD47 gene is knocked in, and the CIITA gene is knocked out.

9. The method of preparing a low immunogenicity pluripotent stem cell according to claim 7 or 8, wherein the donor template comprises a first donor template and a second donor template;

10. A low-immunogenicity pluripotent stem cell, wherein the CIITA gene and the B2M gene of the low-immunogenicity pluripotent stem cell are inactivated and carry a homozygous B2M-HLA E expression cassette and a homozygous CD47 expression cassette.