WO2025217892A1 - Gene editing method for simultaneous knock-in of dna fragments at multiple sites and application thereof - Google Patents

Abstract

Provided are a gene editing method for the simultaneous knock-in of DNA fragments at multiple sites and an application thereof. The method comprises the following steps: making a plurality of double-stranded DNA nicks in a target gene; designing a plurality of homology-directed repair templates; designing exogenous DNA fragments on the homology-directed repair templates; constructing the plurality of homology-directed repair (HDR) templates on the same vector in series; and respectively enabling knock-in of the exogenous DNA fragments into the double-stranded DNA nicks by means of the vector. The multi-site multi-fragment gene knock-in provided by this gene editing method can bring a plurality of DNA fragments into a single vector by means of HDR and enable simultaneous and precise insertion of a plurality of exogenous genes into a target genome at distinct sites, thereby greatly reducing gene editing costs and also increasing the editing efficiency. The gene editing method can be used for basic research and clinical application research.

Description

Gene editing method for simultaneously knocking in DNA fragments at multiple sites and its application Technical Field

The present invention relates to the technical field of biological cell gene editing, and in particular to a gene editing method for simultaneously knocking in DNA fragments at multiple sites.

Background Art

With the rapid development of gene editing technology, technologies such as CRISPR/Cas9 have been widely used in biomedical research, bioengineering, and agriculture as efficient and precise gene editing tools. In many cases, it is necessary to use gene editing technology to edit multiple gene sites at the same time, or to knock in multiple DNA fragments into a gene at the same time to achieve more complex gene regulation or functional modification. For example, in disease treatment and gene therapy, for complex diseases or diseases related to multiple genes, it is necessary to regulate multiple genes at the same time to achieve better therapeutic effects. In addition, in the cultivation of engineered strains and transgenic plants, it is often necessary to introduce multiple exogenous DNA fragments into the genome at the same time to achieve specific functional modifications.

However, although gene editing technologies such as CRISPR/Cas9 can achieve editing at multiple sites, they usually require multiple edits or multiple editing events to complete, and the operation is complicated and inefficient. In addition, for the need to insert multiple DNA fragments at the same time, current gene editing technologies have not yet been able to provide an efficient and simple solution. Therefore, the development of a gene editing method that can achieve the simultaneous knock-in of DNA fragments at multiple sites or the simultaneous modification of multiple genes has important theoretical significance and practical application value. The successful development of this method will expand new possibilities for the further development and application of gene editing technology, and provide strong technical support for innovation in biomedical research, bioengineering and agriculture.

SUMMARY OF THE INVENTION

Technical issues

Currently, multiple exogenous genes are inserted into cells one by one, using a single gene in sequence. For example, lentiviruses are used to insert exogenous genes, or CRISPR/Cas technology is used to create one or more double-stranded DNA cuts, followed by the use of a single AAV virus, ssDNA, or other vector to sequentially insert multiple exogenous genes. However, this method not only involves cumbersome gene knockout and knock-in procedures, but also increases the cost of engineered cell preparation and affects production efficiency.

Technical Solutions

To solve the above technical problems, the invention discloses an efficient, simple-to-operate and low-cost knock-in technology for simultaneously knocking in multiple genes at multiple sites; after creating double-stranded DNA incisions through gene editing technologies such as CRISPR/Cas, multiple exogenous target gene fragments are constructed in series on the same vector; through HDR (homologous recombination-mediated repair), they are accurately inserted into different specific incision sites of the genome at one time; the vector can be provided by AAV, plasmid, PCR amplification fragment, single-stranded DNA or other DNA fragments; and the vector can deliver exogenous genes into cells through delivery methods such as electroporation, viruses or LNP.

The technical solution to the problem to be solved by the present invention is as follows:

A gene editing method for simultaneously knocking in DNA fragments at multiple sites, characterized by comprising the following steps:

Using CRISPR/Cas gene editing technology to create multiple double-stranded DNA cuts in the target gene;

Design a homology-directed repair template with the same number of nicks as the double-stranded DNA;

Designing a foreign DNA fragment on each of the homology-directed repair templates;

All of the homology-directed repair templates designed with exogenous DNA fragments are constructed in tandem on the same vector;

Through the vector and by utilizing the homologous recombination DNA repair method, each of the exogenous DNA fragments is knocked into the corresponding double-stranded DNA nick in the target gene, thereby achieving simultaneous gene knock-in of multiple sites and multiple DNA fragments.

In the above gene editing method, the target genes include TRAC gene, TCR response gene and TGFBR2 receptor gene.

In the above-mentioned gene editing method, the TCR response genes include one or more of PD-1, 41-BB, and IL-2.

In the above-mentioned gene editing method, when using CRISPR/Cas to create a double-stranded DNA nick in the target gene, the following steps are also included:

Based on the nucleic acid sequence of the target gene, use CRISPR/Cas gene editing technology to design two or more gRNA nucleic acid sequences respectively;

Under the action of CRISPR/Cas, the corresponding gRNA sites on the target gene are knocked out through the designed gRNA nucleic acid sequence, creating double-stranded DNA incisions consistent with the number of gRNA nucleic acid sequences.

In the above gene editing method, the gRNA site is a 5’UTR site, a 5’UTR to 3’UTR site, or a 3’UTR site.

In the above-mentioned gene editing method, the exogenous DNA fragment includes one or more of a CAR gene, a cytokine gene, and a functional protein; wherein the cytokine gene includes one or two of IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21 genes; the functional protein includes a fluorescent protein and/or an antibody, etc.

In the above gene editing method, the homology-directed repair template design also includes the following steps:

According to the designed gRNA nucleic acid sequence, upstream homologous nucleic acid sequences and downstream homologous nucleic acid sequences that can carry homologous recombination-mediated repair are designed at both ends of each gRNA nucleic acid sequence to construct a homologous-mediated repair template.

In the above gene editing method, when each homology-directed repair template is designed as an exogenous DNA fragment, the method further includes the following steps:

Based on the nucleic acid sequences on the left and right sides of each double-stranded DNA incision, CRISPR/Cas gene editing technology is used to design upstream homologous nucleic acid sequences and downstream homologous nucleic acid sequences at both ends of each exogenous DNA fragment that can carry homologous recombination-mediated repair.

In the above gene editing method, when each of the exogenous DNA fragments is inserted into the corresponding double-stranded DNA nick via the vector, the following steps are further included:

According to the nucleic acid sequence of the double-stranded DNA nick, each exogenous DNA fragment connected in series on the same vector uses different regions of the vector as homologous-mediated repair templates, and based on the upstream homologous nucleic acid sequence and downstream homologous nucleic acid sequence carried by each for homologous recombination-mediated repair, the exogenous DNA fragments are respectively inserted into the corresponding double-stranded DNA nick.

In the above-mentioned gene editing method, the vector includes one of adeno-associated virus, plasmid, PCR amplicon, and ssDNA.

In the above gene editing method, when the vector knocks each of the exogenous DNA fragments into the corresponding double-stranded DNA nick, the delivery method of the vector to deliver the exogenous DNA fragments is electroporation, virus or LNP cells.

In the above-mentioned gene editing method, the exogenous DNA fragment is inserted into the delivery system of the double-stranded DNA incision through the vector, and an inhibitor is added; for example, the inhibitor is one or more of a DNA-PK inhibitor, RS-1 and L755507; wherein the inhibitor is a DNA-PK inhibitor including one or two of AZD7648 and M3814.

The gene sequences or gene fragments obtained by the above-mentioned gene editing methods, or engineered cells, can be used to prepare drugs or drug components for treating and/or preventing tumors and cancer.

Beneficial effects

The present invention provides an efficient multi-site, multi-fragment gene knock-in method, which can accurately insert multiple DNA fragments into different sites of the genome at one time in an HDR manner, thereby reducing the cost of gene editing while increasing the efficiency of gene editing.

The focus of this invention is to connect DNA repair templates at different sites into a DNA sequence in series, so as to achieve the purpose of one-time delivery. This technology can be applied to any cell and any site to knock in multiple DNA fragments based on the HDR principle, thereby simplifying the gene editing steps and improving the efficiency of gene knock-in.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the gene editing process provided by the present invention;

FIG2 is a schematic diagram of the design of a homology-mediated repair template for inserting the exogenous gene IL-15-GFP;

FIG3 is a schematic diagram of the design of a homology-mediated repair template for inserting exogenous genes IL-15-GFP and CAR through an incision;

FIG4 is a schematic diagram of the design of a homology-mediated repair template for inserting exogenous genes IL-18-GFP and CAR at multiple incisions;

Figure 5 is a schematic diagram of the structure of the exogenous gene GFP vector corresponding to the 5'UTR site, the 5'UTR to 3'UTR site, and the 3'UTR site in Example 1;

Figure 6 is a schematic diagram of the structure of the exogenous gene GFP vector corresponding to the 5'UTR to 3'UTR sites in Example 2;

Figure 7 is a schematic diagram of the vector structure in which the single exogenous genes (GFP gene, CAR gene, IL-15 gene and anti-PD-1 gene) corresponding to the 5'UTR site and 3'UTR site in Example 3 constitute their respective DNA fragments;

Figure 8 is a schematic diagram of the vector structure in which multiple exogenous genes (CAR, GFP, RFP, BFP, YFP, IL-18, IL-10, anti-CTLA4 and other genes) corresponding to the 5'UTR site and 3'UTR site in Example 4 are combined to form multiple DNA fragments;

Figure 9 is a schematic diagram of the vector structure in which multiple exogenous genes corresponding to the 5'UTR to 3'UTR and 3'UTR sites respectively constitute multiple DNA fragments in Example 5;

FIG10 is a flow cytometry diagram showing gene knockout of the PD-1 endogenous gene in T cells of Example 6;

FIG11 is a flow cytometry diagram showing gene knockout of the IL-2 endogenous gene in T cells of Example 6;

FIG12 is a flow cytometry diagram showing gene knockout of the TRAC endogenous gene in T cells of Example 6;

FIG13 is a flow cytometry diagram showing gene knockout of the PD-1 endogenous gene in T cells of Example 6;

Figure 14 is a flow cytometry analysis of the knock-in expression of the exogenous gene CAR in 7-CAR-T cells of Example 6;

Figure 15 is a flow cytometry analysis of the knock-in expression of the exogenous gene IL-12 in 7-CAR-T cells of Example 6;

Figure 16 is a flow cytometry analysis of the knock-in expression of the exogenous gene GFP in 7-CAR-T cells in Example 6;

Figure 17 is a flow cytometry analysis of the knock-in expression of the exogenous gene CAR in 8-CAR-T cells in Example 6;

Figure 18 is a flow cytometry analysis of the knock-in expression of the exogenous gene IL-18 in 8-CAR-T cells of Example 6;

FIG19 is a graph showing the growth and expansion detection of four cell types in Example 6;

Figure 20 is a flow cytometry graph showing the NT cell CAR positivity rate in Example 6;

Figure 21 is a flow cytometry graph showing the CAR positivity rate of Nectin4-CAR-T cells in Example 6;

Figure 22 is a flow cytometry graph showing the CAR positivity rate of 7-CAR-T cells in Example 6;

Figure 23 is a flow cytometry graph showing the CAR positivity rate of 8-CAR-T cells in Example 6;

FIG24 is a flow cytometry image of the expression of NT cells in Example 6;

Figure 25 is a flow cytometry analysis of the expression of Nectin4-CAR-T cells in Example 6;

Figure 26 is a flow cytometry analysis of the knock-in expression of two exogenous genes, IL-12 and GFP, in 7-CAR-T cells in Example 6;

Figure 27 is a flow cytometry analysis of the knock-in expression of the exogenous gene IL-18 in 8-CAR-T cells of Example 6;

FIG28 is a graph showing the specific tumor killing rates of the four types of T cells at an effector-target ratio (E:T=1:1) in Example 6;

Figure 29 is a flow cytometry diagram showing knock-in expression of the exogenous gene CAR in T cells in Example 7;

Figure 30 is a flow cytometry diagram showing knock-in expression of the exogenous gene CAR in NK cells in Example 7;

Figure 31 is a flow cytometry image of the knock-in expression of the exogenous gene CAR in CIK cells in Example 7;

Figure 32 is a flow cytometry analysis of the knock-in expression of the exogenous gene CAR in DC cells of Example 7;

Figure 33 is a flow cytometry diagram showing knock-in expression of the exogenous gene CAR in macrophages of Example 7;

FIG34 is a graph showing growth and expansion detection of 10 cell types in Example 7;

FIG35 is a graph showing the specific tumor killing rate of NT and 7-CAR-T cells at an effector-target ratio (E:T=1:1) in Example 7;

FIG36 is a graph showing the specific tumor killing rate of NK and 7-CAR-NK cells at an effector-target ratio (E:T=1:1) in Example 7;

FIG37 is a graph showing the specific tumor killing rate of CIK and 7-CAR-CIK cells at an effector-target ratio (E:T=1:1) in Example 7;

FIG38 is a graph showing the specific tumor killing rate of DC and 7-CAR-DC cells at an effector-target ratio (E:T=1:1) in Example 7;

Figure 39 is a graph showing the specific tumor killing rate of macrophages (M) and 7-CAR-M at an effector-target ratio (E:T=1:1) in Example 7.

Best Mode for Carrying Out the Invention

The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

The present invention provides a technology that focuses on simultaneous multi-site and multi-gene knock-in, aiming to simultaneously introduce the anti-tumor CAR gene and one or more exogenous genes into corresponding sites on immune cells, such as the 5'UTR site, the 5'UTR to 3'UTR site, or the 3'UTR site; thereby enhancing the anti-tumor activity and persistence of immune cells (such as T cells, NK cells, CD cells, etc.). This method aims to bring revolutionary progress to improving the CAR-immune cell combination therapy for solid tumors.

The present invention utilizes CRISPR/Cas9 gene editing technology to simultaneously transfer multiple segments of gRNA targeting their respective targets into the corresponding gene sites on immune cells through electroporation to create multiple double-strand break incisions (also known as gene site knockouts), and uses a vector carrying CAR and one or more exogenous genes (such as AAV, plasmid or PCR amplicons) as a template to simultaneously insert CAR and one or more exogenous genes into each break incision of the corresponding gene sites of the immune cells.

This multi-site, multi-gene knock-in approach uses a single vector to simultaneously introduce multiple genes, including CARs and exogenous genes, into the gene loci of multiple immune cells. This avoids multiple transduction steps, simplifies the operational process, reduces experimental costs, and enables targeted gene editing at specific sites. This innovative approach has significantly advanced CAR-immune cell engineering technology.

The implementation principle of this invention is:

Multiple DNA fragments repaired with HDR (Homology directed repair, HDR) DNA templates are combined in series, and then delivered to cells through a single vector using DNA delivery technology (such as electroporation, LNP cells or viruses). The cells will use different regions of the DNA fragment as HDR repair templates for DNA repair, thereby achieving the purpose of knocking multiple exogenous DNA fragments into multiple DNA incisions simultaneously with a single vector.

The key points of the present invention are:

1) Gene knock-in method: multiple double-stranded DNA cuts can be created through gene editing technologies such as ZFN, TALENT, and CRISPR/Cas, and then multiple exogenous DNA fragments can be inserted into the corresponding double-stranded DNA cuts through methods such as HDR;

2) HDR repair templates can be delivered by vectors such as AAV, plasmids, PCR amplicons, single-stranded DNA, or other DNA fragments;

3) Exogenous DNA fragments can be inserted into the double-stranded DNA nicks of endogenous genes in cells through delivery methods such as electroporation, viruses, or LNPs;

4) The DNA repair templates at different sites are connected in series to form a DNA sequence, so that a single vector can simultaneously deliver multiple exogenous genes and insert them into different incisions in the endogenous genes of the cell to achieve gene editing.

The gene editing technologies mentioned above may be ZFN, TALENT, and CRISPR/Cas. CRISPR/Cas is preferred in the present invention, which uses CRISPR/Cas to design the gRNA nucleic acid sequence and create multiple double-stranded DNA cuts on the endogenous gene (i.e., target gene) of the cell; wherein the target gene may be one or more of the TRAC gene, TCR response gene, and TGFBR2 receptor gene; and the TCR response gene may include one or more of PD-1, 41-BB, and IL-2.

As shown in FIG1 , the gene editing method for simultaneously knocking in DNA fragments at multiple sites provided by the present invention comprises the following process steps:

S1, using CRISPR/Cas to create multiple double-stranded DNA cuts in the target gene (i.e., endogenous gene);

S2. Design a homology-directed repair template with the same number of nicks as the double-stranded DNA;

S3. Design a foreign DNA fragment on each homology-directed repair template;

S4, multiple homology-directed repair templates are constructed in tandem on the same vector;

S5. Through the vector and the use of homologous recombination DNA repair method, each exogenous DNA fragment is knocked into its corresponding double-stranded DNA incision, achieving simultaneous gene knock-in of multiple sites and multiple DNA fragments.

Step S1 of the above gene editing method further includes the following steps:

S11. Based on the nucleic acid sequence of the target gene, use CRISPR/Cas gene editing technology to design two or more gRNA nucleic acid sequences respectively;

S12. Using CRISPR/Cas gene editing technology and the gRNA nucleic acid sequence designed in step S11, the corresponding gRNA sites on the target gene are knocked out respectively to create double-stranded DNA cuts consistent with the number of gRNA nucleic acid sequences.

In step S1 above, the site of the double-stranded DNA nick, i.e., the gRNA site, can be the 5'UTR site, the 5'UTR to 3'UTR site, or the 3'UTR site. The number of double-stranded DNA nicks can be two, three, or four or more, and can be designed accordingly. Each designed gRNA nucleic acid sequence can produce a corresponding double-stranded DNA nick, and each double-stranded DNA nick can insert a DNA fragment.

In step S2 of the above gene editing method, when designing the homology-directed repair template, the following steps are also included:

According to the gRNA nucleic acid sequence designed in the above step S11, an upstream homologous nucleic acid sequence (LA) and a downstream homologous nucleic acid sequence (RA) capable of carrying homologous recombination-mediated repair are designed at both ends of each gRNA nucleic acid sequence to construct a homologous-mediated repair template.

The number of homology-mediated repair templates is the same as the number of double-stranded DNA cuts to ensure that one double-stranded DNA cut matches one homology-mediated repair template, and the exogenous gene or exogenous DNA fragment is inserted into the double-stranded DNA cut site through the homology-mediated repair template.

In step S3 of the above gene editing method, when designing an exogenous DNA fragment on each of the homology-directed repair templates, the following steps are also included:

According to step S12, the nucleic acid sequences on the left and right sides of the double-stranded DNA nick are obtained, and upstream homologous nucleic acid sequences (LA) and downstream homologous nucleic acid sequences (RA) capable of carrying homologous recombination-mediated repair are designed at both ends of each exogenous DNA fragment.

In the present invention, the upstream homologous nucleic acid sequence (LA) and the downstream homologous nucleic acid sequence (RA) of the homologous recombination-mediated repair template may also be referred to as the left and right homology arms (LA, RA).

Among them, the exogenous DNA fragment includes one or more of the CAR gene, cytokine gene and functional protein; the cytokine gene can include one or more of the IL-7 gene, IL-10 gene, IL-12 gene, IL-15 gene, IL-18 gene, and IL-21 gene; the functional protein can include a fluorescent protein and/or an antibody, and the fluorescent protein includes one or more of the GFP, RFP, BFP and YFP genes; the antibody includes one or more of anti-PD1, anti-CTLA4, anti-TIGIT, anti-TIM3, and anti-LAG3. The left and right homologous arms (LA, RA) at both ends of each exogenous DNA fragment are respectively the same as the left and right homologous arms (LA, RA) of the corresponding homologous recombination-mediated repair template, that is, the corresponding nucleic acid sequences are the same.

In order to facilitate the observation, tracking, and verification of the knock-in rate of cytokines, the exogenous gene GFP fluorescent protein gene (also a type of exogenous gene) can also be designed behind other exogenous genes in the DNA fragment, such as IL-15-GFP, IL-18-GFP, etc., which can be knocked into the double-linked DNA incision of the target gene as exogenous genes.

Step S4 of the above gene editing method further includes the following steps:

The homologous-mediated repair template obtained in the above step S2 and the adjacent homologous recombination-mediated repair template are constructed on the same vector in an alternating head-to-tail manner, such as upstream homologous nucleic acid sequence (LA), downstream homologous nucleic acid sequence (RA), upstream homologous nucleic acid sequence (LA), and downstream homologous nucleic acid sequence (RA).

Alternating end-to-end concatenation, such as the "- LA-A1-RA - LA-A2-RA - LA-A3-RA -" pattern, is used. A1, A2, and A3 represent three exogenous DNA segments, each designed onto three different homologous recombination-mediated repair templates, such as "- LA-A1-RA- ,"" - LA-A2-RA-, " and " - LA-A3-RA- ." Each DNA segment can contain one or more genes or gene expression cassettes. For example, A1 could include a single exogenous gene, CAR, IL-15, or GFP, or multiple exogenous genes, such as CAR-IL-15, IL-15-GFP, or CAR-IL-18-GFP. In other words, a single exogenous DNA segment can contain either one or multiple exogenous gene expression cassettes. However, a single homologous recombination-mediated repair template can correspond to only one DNA segment, and a single DNA segment can only be inserted into one double-stranded DNA nick.

In step S5 of the above gene editing method, when each of the exogenous DNA fragments is inserted into the corresponding double-stranded DNA nick through the vector, the following steps are also included:

According to the nucleic acid sequences on the left and right sides of the double-stranded DNA incision, each exogenous DNA fragment connected in series on the same vector uses different regions of the vector as homologous-mediated repair templates, and based on the upstream homologous nucleic acid sequence (LA) and downstream homologous nucleic acid sequence (RA) carried by each for homologous recombination-mediated repair, the exogenous DNA fragments are inserted into their corresponding double-stranded DNA incisions.

In the above step S5, the vector used can be any one of adeno-associated virus, plasmid, PCR amplicon, and ssDNA, and when the vector knocks each exogenous DNA fragment into the corresponding double-stranded DNA nick, the delivery method of the vector to deliver the exogenous DNA fragment is electroporation, virus or LNP cell.

When constructing the vector, the homology-mediated repair template is constructed into adeno-associated virus AAV, plasmid, PCR amplicon, ssDNA vector, and exogenous DNA fragments are delivered using delivery methods such as electroporation, virus or LNP cells.

In the delivery system where the vector inserts the exogenous DNA fragment into the double-stranded DNA nick, an inhibitor can also be added to inhibit the self-repair of the double-stranded DNA nick and improve the efficiency of the introduction of the exogenous DNA fragment (i.e., the knock-in rate). The inhibitor is one or more of a DNA-PK inhibitor, RS-1, and L755507; wherein the DNA-PK inhibitor includes one or both of AZD7648 and M3814.

The gene editing method for simultaneously knocking in DNA fragments at multiple sites provided by the present invention can be applied to gene editing in various cells, especially gene editing in immune cells, such as T cells, NK cells, CIK cells, DC cells, macrophages, etc.

Before implementing the gene editing method of the present invention for simultaneously knocking in DNA fragments at multiple sites, the following preparations are made.

1. gRNA nucleic acid sequence design

In the present invention, multiple gRNA nucleic acid sequences of the target gene are designed through CRISPR/Cas gene editing technology, and then each gRNA nucleic acid sequence is used to make a corresponding double-stranded DNA cut in the target gene.

Taking target genes such as TRAC gene, PD-1 gene (TCR response gene), 41-BB gene (TCR response gene), IL-2 gene (exogenous cytokine), and TGFBR2 receptor gene (protein) as examples, the corresponding gRNA nucleic acid sequences are designed as follows. Three gRNA nucleic acid sequences are designed for each target gene. Each gRNA nucleic acid sequence can make a corresponding DNA incision. When making the incision, only one gRNA nucleic acid sequence needs to be selected for gene knockout. The specific gRNA nucleic acid sequences are as follows.

(1) The gRNA sequence corresponding to the TRAC gene is any of the following groups:

1.1. The nucleic acid sequence of TRAC-gRNA1 is shown in SEQ ID NO. 1: CCAGCTGCTCGTGATGGACTGGG. Its gene editing site corresponds to the 5’UTR site of the TRAC gene.

1.2. The nucleic acid sequence of TRAC-gRNA2 is shown in SEQ ID NO. 2: TGTACCAGCTGAGAGACTCTCGG. Its gene editing site corresponds to the 5’UTR to 3’UTR site of the TRAC gene.

1.3. The nucleic acid sequence of TRAC-gRNA3 is shown in SEQ ID NO. 3: AGCAAGACGACTGGGGACCCTGG, and its gene editing site corresponds to the 3’UTR site of the TRAC gene;

(2) The nucleic acid sequence of gRNA1 corresponding to PD-1 in the TCR response gene is any of the following groups:

1.4. The nucleic acid sequence of PD-1-gRNA1 is shown in SEQ ID NO. 4: GGAGAAGGCGGCACTCTGGTGGG, and its gene editing site corresponds to the 5’UTR site of the PD-1 gene;

1.5. The nucleic acid sequence of PD-1-gRNA2 is shown in SEQ ID NO. 5: CCGGGCTGGCTGCGGTCCTCGGG. Its gene editing site corresponds to the 5’UTR to 3’UTR site of the PD-1 gene.

1.6. The nucleic acid sequence of PD-1-gRNA3 is shown in SEQ ID NO. 6: AGACCCTCCACCATGAGCCCGGG, and its gene editing site corresponds to the 3’UTR site of the PD-1 gene;

(III) The nucleic acid sequence of gRNA1 corresponding to IL-2 in the TCR response gene is any of the following groups:

1.7. The nucleic acid sequence of IL-2-gRNA1 is shown in SEQ ID NO. 7: ACCCCCAAAGACTGACTGAATGG, and its gene editing site corresponds to the 5’UTR site of the IL-2 gene;

1.8. The nucleic acid sequence of IL-2-gRNA2 is shown in SEQ ID NO. 8: GATTTACAGATGATTTTGAATGG, and its gene editing site corresponds to the 5'UTR to 3'UTR site of the IL-2 gene;

1.9. The nucleic acid sequence of IL-2-gRNA3 is shown in SEQ ID NO.9: AATATAGTATCTAGTAGATTGG, and its gene editing site corresponds to the 3’UTR site of the IL-2 gene.

(IV) The nucleic acid sequence of gRNA1 corresponding to the TGFBR2 receptor gene is any of the following groups:

1.10. The nucleic acid sequence of TGFBR2-gRNA1 is shown in SEQ ID NO. 10: ACTTCAACTCAGCGCTGCGGGGG, and its gene editing site corresponds to the 5’UTR site of the TGFBR2 gene;

1.11. The nucleic acid sequence of TGFBR2-gRNA2 is shown in SEQ ID NO. 11: TGCTGGCGATACGCGTCCACAGG, and its gene editing site corresponds to the 5'UTR to 3'UTR site of the TGFBR2 gene;

1.12. The nucleic acid sequence of TGFBR2-gRNA3 is shown in SEQ ID NO. 12: TGCTTATCCCCACAGCTTACAGG, and its gene editing site corresponds to the 3’UTR site of the TGFBR2 gene;

(V) The nucleic acid sequence of gRNA1 corresponding to 41-BB in the TCR response gene is any of the following:

1.13. The nucleic acid sequence of 41-BB-gRNA1 is shown in SEQ ID NO. 13: GGAGAAGGCGGCACTCTGGTGGG, and its gene editing site corresponds to the 5’UTR site of the 41-BB gene;

1.14. The nucleic acid sequence of 41-BB-gRNA2 is shown in SEQ ID NO. 14: CCGGGCTGGCTGCGGTCCTCGGG. Its gene editing site corresponds to the 5'UTR to 3'UTR site of the 41-BB gene.

1.15. The nucleic acid sequence of 41-BB-gRNA3 is shown in SEQ ID NO.15: AGACCCTCCACCATGAGCCCGGG, and its gene editing site corresponds to the 3’UTR site of the 41-BB gene.

2. Homology-Directed Repair Template Design

For the above-designed gRNA nucleic acid sequence, a homology-mediated repair template is designed, that is, each gRNA nucleic acid sequence corresponds to the designed upstream and downstream homologous nucleic acid sequences (LA/RA) (i.e., left and right homology arms) as any of the following groups.

2.1. The LA and RA sequences corresponding to TRAC-gRNA1 are shown in SEQ ID NO.51 and SEQ ID NO.52 respectively:

2.2, the LA and RA sequences corresponding to TRAC-gRNA2 are shown in SEQ ID NO.53 and SEQ ID NO.54, respectively;

2.3, the LA and RA sequences corresponding to TRAC-gRNA3 are shown in SEQ ID NO.55 and SEQ ID NO.56, respectively;

2.4. The LA and RA sequences corresponding to PD-1-gRNA1 are shown in SEQ ID NO.57 and SEQ ID NO.58, respectively;

2.5. The LA and RA sequences corresponding to PD-1-gRNA2 are shown in SEQ ID NO.59 and SEQ ID NO.60, respectively;

2.6. The LA and RA sequences corresponding to PD-1-gRNA3 are shown in SEQ ID NO.61 and SEQ ID NO.62, respectively;

2.7, IL-2-gRNA1 corresponding to LA and RA sequences are shown in SEQ ID NO. 63 and SEQ ID NO. 64, respectively;

2.8, IL-2-gRNA2 corresponding to LA and RA sequences are shown in SEQ ID NO.65 and SEQ ID NO.66, respectively;

2.9, IL-2-gRNA3 corresponding to LA and RA sequences are shown in SEQ ID NO. 67 and SEQ ID NO. 68, respectively;

2.10, the LA and RA sequences corresponding to TGFBR2-gRNA1 are shown in SEQ ID NO.69 and SEQ ID NO.70, respectively;

2.11. The LA and RA sequences corresponding to TGFBR2-gRNA2 are shown in SEQ ID NO.71 and SEQ ID NO.72, respectively;

2.12. The LA and RA sequences corresponding to TGFBR2-gRNA3 are shown in SEQ ID NO.73 and SEQ ID NO.74, respectively:

The LA and RA sequences corresponding to 2.13 and 41-BB-gRNA1 are shown in SEQ ID NO.75 and SEQ ID NO.76, respectively:

The LA and RA sequences corresponding to 2.14 and 41-BB-gRNA2 are shown in SEQ ID NO.77 and SEQ ID NO.78, respectively:

The LA and RA sequences corresponding to 2.15 and 41-BB-gRNA3 are shown in SEQ ID NO.79 and SEQ ID NO.80, respectively:

3. Design of upstream and downstream homologous nucleic acid sequences of exogenous genes

As shown in Figures 2 and 4, since exogenous genes, such as CAR or IL-15, are activated by the promoter of the target gene (endogenous gene), regulatory elements are added in front of the exogenous gene, such as the T2A regulatory element added in front of CAR and the IRES regulatory element added to the front of other exogenous genes. Using the principle of homologous recombination, left and right homologous arms (LA, RA) with the same nucleic acid sequences as the left and right sides of the double-stranded incision site are designed at both ends of the exogenous gene, and an exogenous DNA fragment is inserted into the double-stranded incision to achieve gene editing of multiple sites simultaneously knocking in DNA fragments. Therefore, the upstream and downstream homologous nucleic acid sequences of the exogenous gene are respectively the same as the upstream and downstream homologous nucleic acid sequences of the corresponding homology-mediated repair template; in this way, the homology-mediated repair template can achieve the tandem insertion of exogenous genes.

As shown in Figure 2 , a double-stranded nick was used to insert two exogenous genes, IL-15-GFP: the upstream homologous nucleic acid sequence of the target gene (LA), the exogenous DNA fragment (IL-15-GFP), and the downstream homologous nucleic acid sequence of the target gene (RA);

As shown in Figure 3 , multiple exogenous genes IL-15-GFP and CAR were inserted into one incision: the upstream homologous nucleic acid sequence of the target gene (LA), the exogenous DNA fragment (IL-15-GFP-CAR), and the downstream homologous nucleic acid sequence of the target gene (RA);

As shown in Figure 4, multiple incisions were made to insert multiple exogenous genes IL-18-GFP and CAR: upstream homologous nucleic acid sequence of the target gene (LA), exogenous DNA fragment (IL-18-GFP), downstream homologous nucleic acid sequence of the target gene (RA), exogenous DNA fragment (CAR), upstream homologous nucleic acid sequence of the target gene (LA), and downstream homologous nucleic acid sequence of the target gene (RA).

Modes for Carrying Out the Invention

The technical solution of the present invention is further described in detail below through some specific implementations.

(1) Gene expression verification

Example 1

The purpose of this example is to use CRISPR/Cas9 gene editing technology to design gRNA nucleic acid sequences corresponding to different target genes to create DNA incisions at different sites to verify whether the genes in each exogenous DNA fragment are expressed.

In this embodiment, the vector is AAV; the endogenous genes are the TRAC gene, PD-1 gene, IL-2 gene and TGFBR2 gene of T cells, the incision sites are the 5'UTR site, the 5'UTR to 3'UTR site and the 3'UTR site; the exogenous gene is the fluorescent protein GFP gene.

1.1 T cell acquisition and activation

Mononuclear cells were isolated from donor peripheral blood using the ficol separation technique and subjected to density gradient centrifugation. T cells were enriched using a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043). T cells were activated, cultured, and expanded using magnetic beads coupled to anti-CD3/anti-CD28. The culture medium used was X-VIVO medium containing 10% FBS and 300 IU/ml rh-IL-2. All T cells were cultured in a 37°C, 5% _CO2 incubator to obtain activated T cells for later use.

1.2 Design of gRNA nucleic acid sequence and GFP structure

1.2.1. Nucleic acid sequence design of endogenous gene gRNA

Prepare 12 groups of T cells. According to the 5’UTR site, 5’UTR to 3’UTR site and 3’UTR site on the TRAC gene, PD-1 gene, IL-2 gene and TGFBR2 gene of each group of T cells, the corresponding nucleic acid sequences of the gRNAs were designed as shown in SEQ ID NO.1 to SEQ ID NO.12 above.

1.2.2. Design of exogenous DNA fragments

As shown in Figure 5, since the GFP structure of the exogenous gene insertion is initiated by the promoter of the endogenous TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene, and is inserted at the 5'UTR site, 5'UTR to 3'UTR site, and 3'UTR site, respectively; therefore, T2A or IRES regulatory elements are added in front of GFP for different regions; using the principle of homologous recombination, left and right homologous arm (LA, RA) nucleic acid series identical to the corresponding incision site nucleic acid sequence are designed at both ends of the GFP gene fragment, respectively, to obtain an exogenous DNA fragment structure containing the GFP gene, namely, the GFP homology-mediated repair template.

According to the 12 groups of gRNA nucleic acid sequences designed in step 1.2.1 of this embodiment, the nucleic acid sequences of the corresponding homology-mediated repair templates (LA, RA) are shown in SEQ ID NO.51 to SEQ ID NO.74 above.

1.3. Making double-stranded DNA cuts

By electroporation, the 5'UTR site, 5'UTR to 3'UTR site, and 3'UTR site on the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene in 12 groups of T cells were knocked out respectively, and the corresponding double-stranded DNA cuts were made, as follows:

For 12 groups of T cells, preheat DPBS in a 37°C incubator and mix it with the complete culture medium of 12 groups of T cells. Then, demagnetize the beads of each activated T cell suspension.

After removing the magnetic beads, count the cells in each group and centrifuge at 500 g for 5 min. Discard the supernatant and resuspend the T cell pellet in 1 mL of 2% FBS + DPBS to obtain a cell suspension. Place the cell suspension in a 37°C incubator for later use.

After incubating the RNP system for 15 minutes at room temperature, the demagnetized T cell suspension of each group was removed from the 37°C incubator and transferred to a centrifuge tube. The suspension was centrifuged at 100 g for 10 minutes. After centrifugation, the supernatant was discarded, and Lonza electroporation buffer was added to each cell pellet at a volume of 2×10 ⁶ cells/20 μL. The composition of the Lonza electroporation buffer (Supplement 1:P3 cell line solution=1:4.5) is shown in Table 1.

Table 1 Lonza electroporation buffer components

Twelve groups of 20 μL of cell suspension were mixed with 2.9 μL of the incubated RNP system to obtain 12 groups of cell-RNP mixtures. Each group of cell-RNP mixtures was then transferred to 12 groups of 16-well electroporation wells for electroporation. After electroporation, 80 μL of preheated DPBS was immediately added to each electroporation well, and the 16-well electroporation plate was transferred to a 37°C incubator and incubated for 15 minutes. Knockout of the 5'UTR site, 5'UTR to 3'UTR site, and 3'UTR site on the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene in 12 groups of T cells was achieved (see Table 3 for details). The composition of the RNP electroporation system is shown in Table 2.

Table 2 RNP electroporation system components

The reagents "TRAC-gRNA, PD-1-gRNA, IL-2-gRNA or TGFBR2-gRNA" in Table 2 are a general term for the gRNA corresponding to each endogenous gene, which can represent gRNA1, gRNA2 or gRNA3, respectively corresponding to the gRNA in Table 3 of this example; therefore, Table 6 actually corresponds to 12 groups of RNP electroporation system components.

1.4. Insertion of exogenous GFP gene

The 12 sets of GFP homology-mediated repair templates designed in step 1.2.2 of this example were constructed into 12 sets of AAV vectors, and 12 exogenous DNA fragments were delivered using electroporation.

After electroporation, 20 μl of AAV virus was added to each of the 12 groups of X-VIVO complete culture medium. After mixing the system, the 12 groups of cell suspensions incubated at 37°C were transferred to the 12 groups of RNP electroporation systems. The 12 gene-edited T cells were placed in a 37°C, 5% _CO2 constant temperature incubator for overnight culture. The 12 groups of AAVs were electroporated to insert the 12 GFP gene fragments into the 5'UTR site, 5'UTR to 3'UTR site, and 3'UTR site on the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene in the 12 groups of T cells, respectively, to obtain the corresponding engineered T cells, as shown in Table 3.

The gene expression (knockout rate and knockin rate) in T cells was verified by flow cytometry, and the results are shown in Table 3.

Table 3 Gene expression in engineered T cells

As can be seen from Table 3, through the gene editing method of the present invention, the exogenous DNA fragments can be expressed normally after the DNA incisions created by knocking out different sites (e.g., 5'UTR site, 5'UTR to 3'UTR site, and 3'UTR site) in different endogenous genes (e.g., TRAC gene, PD-1 gene, IL-2 gene, TGFBR2 gene) are inserted into the exogenous DNA fragments.

Example 2

The purpose of this example is to insert exogenous DNA fragments into the double-stranded DNA nick of the target gene through different vectors and different delivery methods to verify whether the gene in each exogenous DNA fragment is expressed.

In this embodiment, the vectors are AAV, plasmid, PCR amplicon, ssDNA, etc.; the endogenous gene is the TRAC gene of T cells, and the cleavage site is the 5'UTR to 3'UTR site; and the exogenous DNA fragment is the fluorescent protein GFP gene.

2.1 T cell acquisition and activation

The contents are the same as those of step 1.1 in Example 1.

2.2 Design of gRNA Nucleic Acid Sequence and GFP Structure

2.2.1. Nucleic acid sequence design of endogenous gene gRNA

Prepare 7 groups of T cells, and design 7 groups of corresponding TRAC-gRNA2 nucleic acid sequences as shown in SEQ ID NO.2 based on the 5’UTR to 3’UTR sites on the TRAC gene of each group of T cells.

2.2.2. Design of the structure of the exogenous DNA fragment containing the GFP gene

As shown in Figure 6, since the exogenously inserted GFP construct is driven by the promoter of the endogenous TRAC gene and inserted between the 5'UTR and 3'UTR sites, a T2A regulatory element was added in front of GFP. Using the principle of homologous recombination, left and right homologous arms (LA and RA) were designed at either end of the GFP gene fragment, corresponding to the corresponding sites. This yielded an exogenous DNA fragment containing the GFP gene, i.e., the GFP homology-directed repair template. The nucleic acid sequences of the seven TRAC-5'UTR-3'UTR-gRNA2 groups corresponding to the homology-directed repair templates (LA and RA) are shown in SEQ ID NO. 53 and SEQ ID NO. 54.

2.3. Double-stranded DNA nicking on T cells

Knockout of the 5'UTR to 3'UTR site of the TRAC gene in 7 groups of T cells was achieved by electroporation as follows:

For each of the 7 groups of T cells, preheat DPBS in a 37°C incubator and mix it with the complete culture medium of each group of T cells. Then, demagnetize the activated T cell suspensions of each group.

After removing the magnetic beads, count the cells in each group and centrifuge at 500 g for 5 min. Discard the supernatant and resuspend the T cell pellet in 1 mL of 2% FBS + DPBS. Place the cell resuspension in a 37°C incubator until ready for use.

After incubating the RNP system for 15 minutes at room temperature, remove the demagnetized T cells from the 37°C incubator, transfer them to a centrifuge tube, and centrifuge at 100g for 10 minutes. After centrifugation, discard the supernatant, and add Lonza electroporation buffer to the cell pellet at a volume of 2× ¹⁰⁶ cells/20μL per group. The components of Lonza electroporation buffer are shown in Table 1.

Seven groups of 20 μL cells were mixed with 3.4 μL of incubated RNP system to obtain seven groups of cell RNP mixtures. Each group of cell RNP mixtures was then transferred to seven groups of 16-well electroporation wells for electroporation. After electroporation, 80 μL of preheated DPBS was immediately added to each electroporation well, and the plate was moved to a 37°C incubator and incubated for 15 minutes together with the 16-well electroporation plate to achieve knockout of the 5’UTR to 3’UTR site on the TRAC gene in the seven groups of T cells. The composition of the RNP electroporation system was tabulated as shown in Table 4.

Table 4 RNP electroporation system component distribution

2.4. Insertion of exogenous GFP gene

In this embodiment, the exogenous GFP gene can be inserted into the DNA incision by AAV vector delivery or LNP (including plasmids, PCR amplicons, ssDNA and other vectors). Depending on the different vectors and delivery methods, the insertion of the exogenous GFP gene can take the following two forms.

2.4.1 AAV vector-mediated exogenous GFP gene insertion

A set of GFP homology-mediated repair templates designed in step 2.2.2 of this example was constructed into an AAV vector, and DNA fragments were delivered using a viral approach, as shown in Table 6.

After electroporation, 20 μl of AAV virus was added to the X-VIVO complete medium. After the system was mixed, the cell suspension incubated at 37°C was transferred to the RNP electroporation system. The gene-edited T cells were placed in a 37°C, 5% _CO2 constant temperature incubator for overnight culture. The GFP gene fragment was inserted into the 5'UTR to 3'UTR site on the TRAC gene in the T cells through AAV to obtain engineered T cells, as shown in Table 6.

2.4.2 Plasmid, PCR amplicon, and ssDNA vector-mediated insertion of exogenous GFP genes

The six sets of GFP homology-mediated repair templates designed in step 2.2.2 of this example were constructed into plasmids, PCR amplicons, ssDNA vectors, and other vectors (including two sets of plasmids, PCR amplicons, and ssDNA vectors), and DNA fragments were delivered by electroporation and LNP cells, respectively, as shown in Table 6.

After electroporation, 20 μl of plasmid, PCR amplicon, and ssDNA vector (including 2 groups of plasmid, PCR amplicon, and ssDNA vector) were added to each of the 6 groups of X-VIVO complete medium, and the mixture was mixed to obtain 6 mixed systems. The 6 groups of cells incubated in a 37°C incubator were transferred into 6 RNP electroporation systems (as shown in Table 9, 2 groups of plasmid, PCR amplicon, and ssDNA). The 6 groups of gene-edited T cells were then placed in a 37°C, 5% _CO2 constant temperature incubator for overnight culture. The exogenous gene GFP fragment was inserted into the 5'UTR to 3'UTR site on the TRAC gene in the T cells by electroporation or LNP to obtain the corresponding engineered T cells, as shown in Table 6.

Table 5 RNP electroporation system components

Table 5 shows the components of three groups of RNP electroporation systems: Group 1 components: PGA, TRAC-gRNA2, plasmid and TrueCut Cas9; Group 2 components: PGA, TRAC-gRNA2, PCR amplicon and TrueCut Cas9; Group 3 components: PGA, TRAC-gRNA2, ssDNA and TrueCut Cas9.

The gene expression (knockout rate and knockin rate) in T cells was verified by flow cytometry, and the results are shown in Table 6.

Table 6 Gene expression in engineered T cells

As can be seen from Table 6, through the gene editing method of the present invention, the exogenous gene GFP can be delivered to the DNA incision through different vectors (e.g., AAV, plasmid, PCR amplicon, ssDNA vector) and can be expressed normally.

Example 3

The purpose of this example is to insert multiple different exogenous DNA fragments into different double-stranded DNA nicking sites on different endogenous genes to verify whether the genes in each exogenous DNA fragment and the endogenous gene are expressed.

In this embodiment, the vector is AAV; the endogenous genes are the TRAC gene and IL-2 gene of T cells, and the incision sites are the 5'UTR site and the 3'UTR site; the exogenous DNA fragments are DNA fragments such as the GFP gene, CAR gene, anti-PD1 and IL-15 gene.

3.1 T cell acquisition and activation

The contents are the same as those of step 1.1 in Example 1.

4.2 Design of gRNA Nucleic Acid Sequence and Exogenous Gene Structure

4.2.1. Nucleic acid sequence design of endogenous gene gRNA

Prepare 8 groups of T cells. Based on the 5'UTR sites and 3'UTR sites on the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene of each group of T cells, the corresponding gRNA nucleic acid sequences are as follows:

1) TRAC gene 5’UTR site, as shown in SEQ ID NO.1;

2) TRAC gene 3’UTR site, as shown in SEQ ID NO.3;

3) PD-1 gene 5’UTR site, as shown in SEQ ID NO.4;

4) PD-1 gene 3’UTR site, as shown in SEQ ID NO.6;

5) IL-2 gene 5'UTR site, as shown in SEQ ID NO.7;

6) IL-2 gene 3'UTR site, as shown in SEQ ID NO.9;

7) TGFBR2 gene 5'UTR site, as shown in SEQ ID NO.10;

8) TGFBR2 gene 3’UTR site, as shown in SEQ ID NO.12.

3.2.2. Exogenous gene structure design

In this embodiment, the exogenous DNA fragments selected are DNA fragments corresponding to the GFP gene, CAR gene, IL-15 gene and anti-PD-1.

As shown in Figure 7, since the single gene structure into which the exogenous DNA fragment is inserted is initiated by the promoter of the endogenous TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene, and is inserted at the 5'UTR site and 3'UTR site, respectively; therefore, T2A or IRES regulatory elements are added in front of the single gene for different regions; in addition, since different exogenous genes need to be inserted into the corresponding double-stranded DNA incisions at one time, more than two exogenous genes need to be connected in series to the vector, and finally, using the principle of homologous recombination, left and right homologous arms (LA, RA) identical to the corresponding sites are designed at both ends of the exogenous gene fragment, respectively, to obtain the corresponding exogenous DNA fragment structures, namely, the homologous-mediated repair templates of the DNA fragments corresponding to the GFP gene, CAR gene, IL-15 gene, and anti-PD-1 gene, respectively.

The 8 sets of gRNA nucleic acid sequences designed according to step 4.2.1 of this example, and the corresponding nucleic acid sequences of the homology-directed repair templates (LA, RA) are shown below:

1) The 5'UTR site of the TRAC gene and the LA/RA homology arms are shown in SEQ ID NOs. 51 and 52, respectively;

2) The 3’UTR site of the TRAC gene and the LA/RA homology arms are shown in SEQ ID NO. 55 and 56, respectively;

3) The 5'UTR site of the PD-1 gene, the LA/RA homology arms are shown as SEQ ID NO. 57 and 58 respectively;

4) The 3'UTR site of the PD-1 gene, the LA/RA homology arms are shown as SEQ ID NO. 61 and 62 respectively;

5) The 5'UTR site of the IL-2 gene and the LA/RA homology arms are shown in SEQ ID NOs. 63 and 64, respectively;

6) The 3'UTR site of the IL-2 gene and the LA/RA homology arms are shown in SEQ ID NOs. 67 and 68, respectively;

7) TGFBR2 gene 5’UTR site, LA/RA homology arms are shown as SEQ ID NO. 69 and 70 respectively;

8) The 3’UTR site of TGFBR2 gene and the LA/RA homology arms are shown in SEQ ID NO.73 and 74 respectively.

3.3. Creating Multiple Double-Stranded DNA Cuts

By electroporation, the 5'UTR and 3'UTR sites of the TRAC, PD-1, IL-2, and TGFBR2 genes in the eight groups of T cells in step 3.2.1 of this example were knocked out, respectively, to create corresponding double-stranded DNA nicks, as follows:

For 8 groups of T cells, preheat DPBS in a 37°C incubator and mix it with 8 groups of T cell complete culture medium. Then, demagnetize the activated T cell suspension of each group.

After removing the magnetic beads, count the cells in each group, centrifuge at 500 g for 5 min, discard the supernatant, resuspend the T cell pellet in each group with 1 mL of 2% FBS + DPBS washing solution, and place the cell resuspension in a 37°C incubator until used;

After incubating the RNP system at room temperature for 15 minutes, the demagnetized T cells were removed from the 37°C incubator and transferred to a centrifuge tube. The tubes were then centrifuged at 100 g for 10 minutes. After centrifugation, the supernatant was discarded, and Lonza electroporation buffer was added to each cell pellet at a concentration of 2 × 10 ⁶ cells/20 μL. The components of the Lonza electroporation buffer are shown in Table 1.

Eight groups of 20 μL cells were mixed with 3.4 μL of incubated RNP system to obtain eight groups of cell-RNP mixtures. Each group of cell-RNP mixtures was then transferred to eight groups of 16-well electroporation wells. After electroporation, 80 μL of preheated DPBS was immediately added to each electroporation well, and the plates were moved to a 37°C incubator and incubated for 15 minutes together with the 16-well electroporation plate. The 5’UTR and 3’UTR sites on the TRAC, PD-1, IL-2, and TGFBR2 genes of the twelve groups of T cells were knocked out (see Table 9 for details). The composition of the RNP electroporation system is shown in Tables 7 and 8.

Table 7 RNP electroporation system components

Table 8 RNP electroporation system components

3.4. Insertion of foreign DNA fragments

The 8 groups of homology-mediated repair templates for 8 exogenous DNA fragments corresponding to the GFP gene, CAR gene, IL-15 gene, and anti-PD-1 gene designed in step 3.2.2 of this example were constructed into 8 groups of AAV vectors, and the 8 exogenous DNA fragments were delivered separately by electroporation.

After electroporation, 20 μl of AAV virus was added to each of the 8 groups of X-VIVO complete culture medium. After mixing the system, the 8 groups of cell suspensions incubated at 37°C were transferred to the 8 groups of RNP electroporation systems. The 8 groups of gene-edited T cells were placed in a 37°C, 5% _CO2 constant temperature incubator for overnight culture. The 8 exogenous genes, such as GFP gene fragment, exogenous CAR gene fragment, exogenous IL-15 gene fragment and exogenous anti-PD-1 fragment, were electroporated into the 5'UTR site and 3'UTR site on the TRAC gene, PD-1 gene, IL-2 gene and TGFBR2 gene of the 8 groups of T cells respectively through 8 groups of AAV to obtain the corresponding engineered T cells (see Table 9 for details).

The gene expression (knockout rate and knock-in rate) in T cells was verified by flow cytometry, and the results are shown in Table 9.

Table 9 Gene expression in engineered T cells

As can be seen from Table 9, through the gene editing method of the present invention, knockout of different sites (e.g., 5'UTR site, 3'UTR site) in different endogenous genes (e.g., TRAC gene, PD-1 gene, IL-2 gene, TGFBR2 gene) can produce multiple DNA incisions; at the same time, after multiple exogenous DNA fragments (e.g., GFP gene fragment, CAR gene fragment, IL-15 gene fragment and anti-PD-1 fragment) are inserted into the corresponding DNA incision sites, each exogenous gene and each endogenous gene can be expressed normally.

Example 4

The purpose of this example is to insert multiple different exogenous DNA fragments into the double-stranded DNA nicking sites on different endogenous genes to verify whether the genes in each exogenous DNA fragment and the endogenous gene are expressed.

In this embodiment, the vector is AAV; the endogenous genes are the TRAC gene, PD-1 gene, IL-2 gene and TGFBR2 gene of T cells, and the DNA sites are the 5'UTR site and the 3'UTR site; the exogenous DNA fragments are fluorescent proteins (such as GFP, RFP, BFP, YFP genes), CAR genes, antibody proteins (anti-PD1, anti-CTLA4) and cytokines (such as IL-10 gene, IL-18 gene) and other DNA fragments.

4.1 T cell acquisition and activation

The contents are the same as those of step 1.1 in Example 1.

4.2 Design of gRNA Nucleic Acid Sequence and Exogenous Gene Structure

4.2.1. Nucleic acid sequence design of endogenous gene gRNA

Prepare 8 groups of T cells. Based on the 5'UTR sites and 3'UTR sites on the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene of each group of T cells, the corresponding gRNA nucleic acid sequences are as follows:

1) TRAC gene 5'UTR site, as shown in SEQ ID NO.1;

2) TRAC gene 3’UTR site, as shown in SEQ ID NO.3;

3) PD-1 gene 5’UTR site, as shown in SEQ ID NO.4;

4) PD-1 gene 3’UTR site, as shown in SEQ ID NO.6;

5) IL-2 gene 5'UTR site, as shown in SEQ ID NO.7;

6) IL-2 gene 3'UTR site, as shown in SEQ ID NO.9;

7) TGFBR2 gene 5'UTR site, as shown in SEQ ID NO.10;

8) TGFBR2 gene 3’UTR site, as shown in SEQ ID NO.12.

4.2.2. Exogenous gene structure design

In this embodiment, the exogenous DNA fragments selected are exogenous DNA fragments corresponding to gene combinations containing CAR, GFP, RFP, BFP, YFP, IL-18, IL-10, anti-CTLA4, etc., such as CAR-RFP gene combination DNA fragment, IL-18-BFP gene combination DNA fragment, IL-10-YFP gene combination DNA fragment and anti-CTLA4-GFP gene combination DNA fragment.

As shown in Figure 8, since the single gene structure inserted by each exogenous gene is initiated by the promoter of the endogenous TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene, and is inserted into the 5'UTR site and 3'UTR site respectively; Therefore, for different regions, T2A or IRES regulatory elements are added in front of the single gene; In addition, since different exogenous genes need to be inserted into the corresponding multiple double-stranded DNA incisions at one time, it is necessary to connect more than two exogenous genes in series to the vector, and finally, using the principle of homologous recombination, the left and right homologous arms (LA, RA) identical to the corresponding sites are designed at both ends of the exogenous gene fragment to obtain the corresponding exogenous DNA fragment structure, that is, the DNA fragment homology-mediated repair template corresponding to each exogenous DNA gene. According to the 8 groups of gRNA nucleic acid sequences designed in step 4.2.1 of this embodiment, the nucleic acid sequences of the corresponding designed homology-mediated repair templates (LA, RA) are as follows:

1) The 5'UTR site of the TRAC gene and the LA/RA homology arms are shown in SEQ ID NOs. 51 and 52, respectively;

2) The 3’UTR site of the TRAC gene and the LA/RA homology arms are shown in SEQ ID NO. 55 and 56, respectively;

3) The 5'UTR site of the PD-1 gene, the LA/RA homology arms are shown as SEQ ID NO. 57 and 58 respectively;

4) The 3'UTR site of the PD-1 gene, the LA/RA homology arms are shown as SEQ ID NO. 61 and 62 respectively;

5) The 5'UTR site of the IL-2 gene and the LA/RA homology arms are shown in SEQ ID NOs. 63 and 64, respectively;

6) The 3'UTR site of the IL-2 gene, the LA/RA homology arms are shown as SEQ ID NO. 67 and 68, respectively;

7) TGFBR2 gene 5’UTR site, LA/RA homology arms are shown as SEQ ID NO. 69 and 70 respectively;

8) The 3’UTR site of TGFBR2 gene and the LA/RA homology arms are shown in SEQ ID NO.73 and 74 respectively.

4.3. Creating Multiple DNA Cuts on T Cells

By electroporation, the 5'UTR sites and 3'UTR sites of the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene of the 8 groups of T cells in step 4.2.1 of this example were respectively modified as follows:

For 8 groups of T cells, preheat DPBS in a 37°C incubator and mix it with the complete culture medium of each group of T cells. Then, demagnetize the beads of each activated T cell suspension.

After removing the magnetic beads, count the cells in each group, centrifuge at 500 g for 5 min, discard the supernatant, resuspend the T cell pellet in each group with 1 mL of 2% FBS + DPBS washing solution, and place the cell resuspension in a 37°C incubator until used;

After incubating the RNP system at room temperature for 15 minutes, the eight groups of T cells with demagnetized beads were removed from the 37°C incubator and transferred to centrifuge tubes. They were then centrifuged at 100 g for 10 minutes. After centrifugation, the supernatant was discarded, and Lonza electroporation buffer was added to each cell pellet at a volume of 2×10 ⁶ cells/20 μL. The components of the Lonza electroporation buffer are shown in Table 1.

Eight groups of 20 μL cells were mixed with 3.4 μL of incubated RNP system to obtain eight groups of cell RNP mixtures, and then each group of cell RNP mixtures was transferred to eight groups of 16-well electroporation wells. After electroporation, preheated 80 μL of DPBS was immediately added to each electroporation well, and the plates were moved to a 37°C incubator and incubated for 15 minutes together with the 16-well electroporation plate. The 5’UTR sites and 3’UTR sites on the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene of the eight groups of T cells were knocked out respectively (see Table 12 for details). Among them, due to the differences between endogenous genes and exogenous DNA fragments, the components of the RNP electroporation system will also be different, as shown in Tables 10 and 11.

Table 10 RNP electroporation system components

Table 11 RNP electroporation system components

4.4. Insertion of foreign genes

The 8 groups of homology-mediated repair templates corresponding to 8 exogenous DNA fragments, each containing fluorescent proteins (e.g., GFP, RFP, BFP, YFP genes), CAR genes, antibody proteins (anti-PD1, anti-CTLA4) and cytokines (e.g., IL-10 gene, IL-18 gene), designed in step 4.2.2 of this example, were constructed into the same 8 groups of AAV vectors, and the 8 exogenous DNA fragments were delivered separately by electroporation.

After electroporation, 20 μl of AAV virus was added to each of the 8 groups of X-VIVO complete culture medium. After mixing the system, the 8 groups of cell suspensions incubated at 37°C were transferred to 8 groups of RNP electroporation systems. The 8 groups of gene-edited T cells were placed in a 37°C, 5% _CO2 constant temperature incubator for overnight culture. The 8 exogenous DNA fragments were inserted into the 5'UTR site and 3'UTR site on the TRAC gene, PD-1 gene, IL-2 gene, and TGFBR2 gene of the T cells by electroporation of the 8 groups of AAV, respectively, to obtain the corresponding engineered T cells (see Table 12 for details).

The gene expression (knockout rate and knock-in rate) in T cells was verified by flow cytometry, and the results are shown in Table 12.

Table 12 Gene expression in engineered T cells

As can be seen from Table 12, through the gene editing method of the present invention, DNA incisions are made at different sites (e.g., 5'UTR site, 3'UTR site) in different endogenous genes (e.g., TRAC gene, PD-1 gene, IL-2 gene, TGFBR2 gene), and DNA fragments containing multiple exogenous genes, such as fluorescent proteins (e.g., GFP, RFP, BFP, YFP genes), CAR genes, antibody proteins (anti-PD1, anti-CTLA4) and cytokines (e.g., IL-10 gene, IL-18 gene), are respectively inserted. After that, each exogenous gene and endogenous gene can be expressed normally.

Example 5

The purpose of this example is to verify whether the inhibitor in the delivery system of the vector for delivering exogenous genes can normally express the exogenous genes and endogenous genes.

In this example, the vector is AAV, and the CAR exogenous gene is inserted into the 5'UTR to 3'UTR site of the TRAC gene on the T cell, and the IL-15+GFP exogenous gene is inserted into the 3'UTR site of the IL-2 gene on the T cell; the differences in gene editing efficiency of different inhibitor combinations are compared;

5.1 T cell acquisition and activation

The contents are the same as those of step 1.1 in Example 1.

5.2 Design of gRNA Nucleic Acid Sequence and IL-15+GFP Structure

5.2.1. Endogenous gene gRNA nucleic acid sequence design

Prepare 9 groups of T cells, and design the nucleic acid sequences of gRNA corresponding to the 5’UTR to 3’UTR sites on the TRAC gene and the 3’UTR sites on the IL-2 gene of the 9 groups of T cells; among them, the nucleic acid sequence corresponding to the 5’UTR to 3’UTR sites of the TRAC gene of the T cells and the 3’UTR sites on the IL-2 gene are as shown in SEQ ID NO.2, and the nucleic acid sequence corresponding to the 3’UTR site of the IL-2-gRNA3 gene is as shown in SEQ ID NO.9.

5.2.2. Design of the exogenous gene CAR and IL-15+GFP structure

As shown in Figure 9, since the CAR structure is initiated by the promoter of endogenous TRAC, a T2A regulatory element is added in front of CAR. In addition, in order to be able to express IL-15+GFP protein while initiating expression of IL-2, this embodiment adds an IRES regulatory element to the front of IL-15+GFP. Since different exogenous genes need to be inserted into the corresponding double-stranded DNA incisions at one time, more than two exogenous genes need to be connected in series to the vector. Finally, using the principle of homologous recombination, left and right homologous arms (LA, RA) identical to the corresponding sites are designed at both ends of the CAR and IL-15+GFP gene fragments, respectively, to obtain the structure of the dual-target gene (CAR, IL-15+GFP), that is, the homologous mediated repair templates corresponding to CAR and IL-15+GFP.

According to the gRNA nucleic acid sequence designed in step 6.2.1 of this embodiment, the nucleic acid sequences of the homology-mediated repair templates (LA, RA) corresponding to TRAC-gRNA2 are shown in SEQ ID NO. 53 and 54, respectively, and the nucleic acid sequences of the homology-mediated repair templates (LA, RA) corresponding to IL-2-gRNA3 are shown in SEQ ID NO. 71 and 72, respectively.

5.3. TRAC and IL-2 genes on T cells create DNA cuts

By electroporation, the 5'UTR to 3'UTR site on the TRAC gene and the 3'UTR site on the IL-2 gene in 9 groups of T cells were knocked out as follows:

For each of the 9 groups of T cells, preheat DPBS in a 37°C incubator and mix it with the complete culture medium of each group of T cells. Then, demagnetize the activated T cell suspension of each group.

After removing the magnetic beads, count the cells in each group, centrifuge at 500 g for 5 min, discard the supernatant, resuspend the T cell pellet in each group with 1 mL of 2% FBS + DPBS washing solution, and place the cell resuspension in a 37°C incubator until used;

After incubating the RNP system at room temperature for 15 minutes, each group of T cells with demagnetized beads was removed from the 37°C incubator and transferred to a centrifuge tube. The cells were centrifuged at 100 g for 10 minutes. After centrifugation, the supernatant was discarded and Lonza electroporation buffer was added to each cell pellet at a volume of 2×10 ⁶ cells/20 μL. The components of the Lonza electroporation buffer are shown in Table 1.

9 groups of 20 μL cells were mixed with 3.4 μL of incubated RNP system to obtain 9 groups of cell RNP mixtures, and then each group of cell RNP mixtures was transferred to 9 groups of 16-well electroporation wells. After electroporation, preheated 80 μL of DPBS was immediately added to each electroporation well, and the 16-well electroporation plate was moved to a 37°C incubator and incubated for 15 minutes; the 5'UTR to 3'UTR site on the TRAC gene and the 3'UTR site on the IL-2 gene on the 4 groups of T cells were knocked out (as shown in Table 14); among them, the composition of the RNP electroporation system was tabulated as shown in Table 14.

Table 13 RNP electroporation system components

Table 13 shows two groups of RNP electroporation system composition formulations: Group 1: PGA, TRAC-gRNA2 and TrueCut Cas9; Group 2: PGA, IL-2-gRNA3 and TrueCut Cas9.

5.4. Insertion of foreign genes

The nine sets of exogenous CAR and IL-15+GFP gene homology-mediated repair templates designed in step 5.2.2 of this example were constructed into the same AAV vector, and DNA fragments were delivered by electroporation.

After electroporation, 20 μl of AAV virus was added to the X-VIVO complete medium to obtain a mixed medium system, and inhibitors (such as AZD7648, M3814 or one or more small molecule compounds such as RS-1 and L755507) were added to the mixed medium system. After mixing the system, the cell suspension incubated in a 37°C incubator was transferred to the RNP electroporation system, and the gene-edited T cells were placed in a 37°C, 5% _CO2 constant temperature incubator for overnight culture. The CAR and IL-15+GFP gene fragments were inserted into the 5'UTR to 3'UTR site on the TRAC gene and the 3'UTR site on the IL-2 gene in the T cells through AAV to obtain engineered T cells, as shown in Table 14.

The gene expression (knockout rate and knock-in rate) in T cells was verified by flow cytometry, and the results are shown in Table 14.

Table 14 Gene expression in engineered T cells

As can be seen from Table 14, after the inhibitor is added to the delivery system during the process of the vector delivering the exogenous gene to the nicking site of the endogenous gene, it can be clearly seen from Groups 2 to 8 that the inhibitor can improve the introduction efficiency of the exogenous DNA fragments, that is, the inhibitor can inhibit the self-repair of the double-stranded DNA nick and improve the introduction efficiency of the exogenous DNA fragments; at the same time, the addition of the inhibitor does not affect the normal expression of the endogenous and exogenous genes.

(II) Functional verification after cell gene editing

Example 6

6.1 Orthogonal Experimental Design of T Cell Gene Editing Methods

This example will use the gene editing methods of T cells in Examples 1 to 5, such as the vector, transduction method, number and type of endogenous genes, gene editing site, number and type of inserted exogenous genes, and type of inhibitors, to design orthogonal combination experiments to demonstrate versatility and verify the function of the prepared T cells; the orthogonal experimental design is shown in Table 15.

Table 15 Orthogonal experimental design table for the preparation of engineered T cells

6.2 Functional Verification of Engineered T Cells

6.2.1. Endogenous gene knockout expression rate and exogenous gene knockin expression rate

The universal verification of the method combination of the present invention was carried out using the design scheme in the orthogonal test Table 15, and the verification results are shown in Table 16.

Taking numbers 7 and 8 in Table 16 as examples, the functions of CAR-T cells prepared by the method of the present invention are described.

The gene sequencing results shown in Figures 10 to 13 show that the gRNAs corresponding to the nucleic acid sequences in numbers 7 and 8 in Table 16 achieved knockout efficiencies of 97.77%, 96.43%, 94.87%, and 97.23% for the endogenous PD-1 gene, the endogenous IL-2 gene, and the endogenous TRAC gene, respectively, in T cells. In other words, the gene knockout efficiencies were all relatively high. Figure 10 shows the endogenous PD-1 gene, Figure 11 shows the endogenous IL-2 gene, Figure 12 shows the endogenous TRAC gene, and Figure 13 shows the endogenous PD-1 gene.

After knocking out the endogenous genes PD-1+IL-2 and TRAC+PD-1 in human immune T cells, the nucleic acid sequences of the schemes shown in No. 7 and 8 in Table 18 were knocked in. The corresponding CAR-T cells in No. 7 and 8 in Table 24 are referred to as 7-CAR-T and 8-CAR-T cells, respectively. The results of the exogenous gene knock-in efficiency are shown in Figures 14 to 18. Figures 14 to 18 are divided into expression rate graphs of knock-in genes of exogenous genes; among them, Figures 14 to 16 respectively represent the knock-in expression rate graphs of the exogenous genes CAR, IL-12, and GFP in No. 7, and Figures 17 to 18 respectively represent the knock-in expression rate graphs of the exogenous genes CAR and IL-18 in No. 8.

6.2.2 T cell function verification

7-CAR-T and 8-CAR-T cells were transferred to 24-well plates respectively and placed in a 37°C, 5% _CO2 constant temperature incubator for further culture until ready for use.

Four types of T cells, including NT cells (blank control), Nectin4-CAR-T cells (positive control cells), 7-CAR-T cells, and 8-CAR-T cells, were cultured separately. Samples were taken on days 5, 7, 9, 11, and 13 to detect cell number, as well as T cell samples to detect CAR positivity, exogenous gene expression, and inserted endogenous gene expression. During the T cell culture process, culture medium was added every 1-2 days for passage. This part is prior art and will not be repeated here.

The expansion test results of the four T cell types are shown in Figure 19. As shown in Figure 19, the expansion of NT cells (blank control) and Nectin4-CAR-T cells is not much different. While electroporation of 7-CAR-T and 8-CAR-T cells has a certain impact on cell expansion, the overall cell expansion can still meet the needs of subsequent experiments.

As can be seen from Figures 20 to 23, the CAR positivity test results of NT cells (blank control), Nectin4-CAR-T cells, 7-CAR-T and 8-CAR-T cells were 0.39%, 92.03%, 88.35% and 85.96%, respectively; therefore, although the expression of the electroporated knock-in CAR gene is lower than that of normal CAR-T cells, its function will not be lost due to electroporation.

As can be seen from Figures 24 to 27, the expression rates of the four types of T cells, such as NT, Nectin4-CAR-T, 7-CAR-T and 8-CAR-T cells, among which 7-CAR-T cells contain two exogenous genes: IL-12 and GFP, and 8-CAR-T cells contain the exogenous gene IL-18, are 45.20%, 48.34% and 50.53%, respectively, which are all better than the expression rates of NT and Nectin4-CAR-T.

The positive rates of the four types of T cells, the expression of exogenous genes, and the expression detection results of the inserted endogenous genes are shown in Table 17.

Table 17 Expression efficiency of endogenous genes after exogenous genes were inserted into T cells

As can be seen from Table 17, after the exogenous CAR and IL-12 genes were inserted into the 5'UTR to 3'UTR sites of the endogenous genes IL-2 or PD-1, respectively, the expression rates of the respective endogenous genes (10% and 9%) were much lower than the expression rates of the respective endogenous genes (99% and 98%) after the exogenous CAR and IL-18 genes were inserted into the 3'UTR sites of the endogenous genes TRAC or PD-1, respectively; this indicates that the insertion of exogenous genes into the 5'UTR to 3'UTR sites will affect the expression of endogenous genes; therefore, when editing T cells, different gene sites should be selected for knockout and insertion according to different uses.

6.2.3. Verification of Tumor Killing Rate of Engineered T Cells

As shown in Figure 28, the specific tumor killing rate curve of the four types of T cells in the effector-target ratio (E:T=1:1) is shown. As shown in Figure 28, the gene-edited 7-CAR-T and 8-CAR-T cells are significantly better than the NT cells and Nectin4-CAR-T cells that have not undergone gene editing in terms of killing efficiency or tumor killing rate. The reason is that the target gene knocked into the IL-12 or IL-18 gene of the immune T cell is induced to express after being stimulated by the target antigen and has a positive effect on the CAR-T cell, so the killing efficiency is better than that of the unedited T cell.

(III) Functional verification of exogenous genes on different immune cells

Example 7

7.1. Verification of endogenous gene knockout rate and exogenous gene knock-in rate;

Taking No. 8 in Table 20 of Example 6 as an example, the application of the method in immune cells, such as T cells, NK cells, CIK cells, DC cells, and macrophages, was performed after knocking out the TRAC gene in these immune cells. The results are shown in Table 18.

Table 18 Gene knockout rate of each immune cell TARC

From the gene knockout efficiency results shown in Table 18, it can be seen that the TRAC gene knockout efficiency of the gRNA corresponding to the nucleic acid sequence for T cells, NK cells, CIK cells, DC cells, and macrophages is 93%, 92%, 90%, 91%, and 89%, respectively, indicating that the gene knockout efficiency is relatively high.

After knocking out the TRAC gene in human immune T cells, NK cells, CIK cells, DC cells, and macrophages, the nucleic acid sequence exogenous gene CAR according to the scheme corresponding to number 8 in Table 18 was knocked in. The nucleic acid sequence gRNA knocked the exogenous gene CAR into T cells, NK cells, CIK cells, DC cells, and macrophages at the corresponding TRAC gene knockout sites. The knock-in efficiency results are shown in Figures 21 to 25.

In Figures 29 to 33, the corresponding knock-in efficiency graphs respectively represent the knock-in efficiency of the exogenous gene CAR on the TRAC gene of T cells, NK cells, CIK cells, DC cells, and macrophages, and the corresponding knock-in efficiencies are 63.14%, 72.72%, 79.97%, 73.16%, and 66.39%, respectively. This shows that the knock-in efficiency of exogenous genes in different immune cells is still relatively high.

7.2. Verification of Immune Cell Expansion and Growth

CAR-T, CAR-NK, CAR-CIK, CAR-DC, and CAR-macrophages were obtained using the scheme corresponding to number 7 in Table 18 of Example 6, which are referred to as 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC, and 7-CAR-M cells, respectively.

7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC, and 7-CAR-M cells were transferred to 24-well plates and placed in a 37°C, 5% _CO2 constant temperature incubator for further culture until ready for use.

Ten cell types, including NT cells, NK cells, CIK cells, DC cells, macrophages, 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC, and 7-CAR-M cells, were cultured separately. Samples were taken on days 5, 7, 9, 11, and 13 to detect cell number, and T cells were sampled to detect CAR positivity, exogenous gene expression, and inserted endogenous gene expression. During the cell culture process, culture medium was added every 1-2 days for passage. This part is prior art and will not be described in detail here.

As shown in Figure 34, the expansion test results of 10 cell types are shown. As shown in Figure 34, NT cells, NK cells, CIK cells, DC cells, and macrophages (blank control) proliferate relatively quickly, while 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC, and 7-CAR-M cells undergo electroporation. Electroporation has a certain effect on cell expansion, but the effect is not significant. 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC, and 7-CAR-M cells also expand, and the overall expansion can meet the needs of subsequent experiments.

7.3. Immune Cell Knockout and Knockin Verification

The immune cells described in Section 7.2 are used, the endogenous gene is PD-1, the exogenous gene is IL-18, and the knockout or insertion site is the 3'UTR site.

As shown in Figure 25, the positive rates, expression of exogenous genes, and expression of inserted endogenous genes of 10 cell types, including NT cells, NK cells, CIK cells, DC cells, macrophages, 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC, and 7-CAR-M, are shown in Table 19.

Table 19 Gene knockout rate of each immune cell TARC

As shown in Table 19, the results of exogenous gene detection in 10 cell types, including NT cells, NK cells, CIK cells, DC cells, macrophages, 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC and 7-CAR-M, showed that the exogenous IL-18 gene knocked in by electroporation can be expressed normally; at the same time, since the exogenous gene is inserted into the 3'UTR position, it does not affect the expression of the endogenous gene PD-1.

7.4 Verification of Tumor Killing Rate of Immune Cells

The immune cells described in Section 7.2 are used, the endogenous gene is PD-1, the exogenous gene is IL-18, and the knockout or insertion site is the 3'UTR site.

[Corrected 06.05.2024 in accordance with Article 91]
As shown in Figures 35 to 39, the specific tumor killing rate curves of 10 types of T cells, including NT cells, NK cells, CIK cells, DC cells, macrophages, 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC and 7-CAR-M, are shown in the effector-target ratio (E:T=1:1); among them, Figure 35 shows the tumor killing rate curve of NT and 7-CAR-T cells, Figure 36 shows the tumor killing rate curve of NK and 7-CAR-NK cells, Figure 37 shows the tumor killing rate curve of DC and 7-CAR-DC cells, Figure 38 shows the tumor killing rate curve of CIK and 7-CAR-CIK cells, and Figure 39 shows the tumor killing rate curve of macrophages and 7-CAR-M cells.

As shown in Figures 35 to 39, gene-edited 7-CAR-T, 7-CAR-NK, 7-CAR-CIK, 7-CAR-DC, and 7-CAR-M cells exhibit significantly better killing efficiency or tumorigenesis rates than unedited NT cells, NK cells, CIK cells, DC cells, and macrophages. This demonstrates that the gene editing technology described in this patent can be applied to various immune cells.

Industrial Applicability

The multi-site, multi-fragment gene knock-in technology provided by the present invention can use a single vector to simultaneously and accurately insert exogenous genes into different sites of the target genome using multiple DNA fragments via HDR (Homology Directed Repair). This gene editing technology reduces the cost of gene editing while increasing editing efficiency, and can be used for basic research and clinical application research.

Claims (18)

A gene editing method for simultaneously knocking in DNA fragments at multiple sites, characterized by comprising the following steps: Using CRISPR/Cas gene editing technology to create multiple double-stranded DNA cuts in the target gene; Designing a homology-directed repair template with the same number of double-stranded DNA nicks; Designing a foreign DNA fragment on each of the homology-directed repair templates; All of the homology-directed repair templates designed with exogenous DNA fragments are constructed in tandem on the same vector; Through the vector and by utilizing the homologous recombination DNA repair method, each of the exogenous DNA fragments is knocked into the corresponding double-stranded DNA nick in the target gene, thereby achieving simultaneous gene knock-in of multiple sites and multiple DNA fragments. The gene editing method according to claim 1, characterized in that the target genes include TRAC genes, TCR response genes and TGFBR2 receptor genes. The gene editing method according to claim 2, characterized in that the TCR response gene includes one or more of PD-1, 41-BB, and IL-2. The gene editing method according to claim 1, characterized in that when using CRISPR/Cas to create a double-stranded DNA nick in the target gene, it also includes the following steps: Based on the nucleic acid sequence of the target gene, use CRISPR/Cas gene editing technology to design two or more gRNA nucleic acid sequences respectively; Under the action of CRISPR/Cas, the corresponding gRNA sites on the target gene are knocked out respectively through the designed gRNA nucleic acid sequence, creating double-stranded DNA incisions consistent with the number of gRNA nucleic acid sequences. The gene editing method according to claim 4, wherein the gRNA site is a 5’UTR site, a 5’UTR to 3’UTR site, or a 3’UTR site. The gene editing method according to claim 4, characterized in that when designing the homology-directed repair template, the following steps are further included: According to the designed gRNA nucleic acid sequence, upstream homologous nucleic acid sequences and downstream homologous nucleic acid sequences that can carry homologous recombination-mediated repair are designed at both ends of each gRNA nucleic acid sequence to construct a homologous-mediated repair template. The gene editing method according to claim 1, characterized in that the exogenous DNA fragment includes one or more of a CAR gene, a cytokine gene, and a functional protein. The gene editing method according to claim 7, characterized in that the cytokine gene includes one or two of the IL-7 gene, IL-10 gene, IL-12 gene, IL-15 gene, IL-18 gene and IL-21 gene; and the functional protein includes a fluorescent protein and/or an antibody. The gene editing method according to claim 8, characterized in that the fluorescent protein includes one or more of GFP, RFP, BFP and YFP genes; the antibody includes one or more of anti-PD1, anti-CTLA4, anti-TIGIT, anti-TIM3, and anti-LAG3. The gene editing method according to claim 1, characterized in that when designing an exogenous DNA fragment on each of the homology-directed repair templates, it further comprises the steps of: According to the nucleic acid sequences on the left and right sides of each double-stranded DNA nick, upstream homologous nucleic acid sequences and downstream homologous nucleic acid sequences capable of carrying homologous recombination-mediated repair are designed at both ends of each exogenous DNA fragment. The gene editing method according to claim 10, characterized in that when each of the exogenous DNA fragments is inserted into the corresponding double-stranded DNA nick through the vector, the method further comprises the following steps: According to the nucleic acid sequences on the left and right sides of the double-stranded DNA incision, each exogenous DNA fragment connected in series on the same vector uses different regions of the vector as homologous-mediated repair templates, and based on the upstream homologous nucleic acid sequences and downstream homologous nucleic acid sequences carried by each for homologous recombination-mediated repair, the exogenous DNA fragments are inserted into the corresponding double-stranded DNA incision. The gene editing method according to claim 1, characterized in that the vector comprises one of an adeno-associated virus, a plasmid, a PCR amplicon, and ssDNA. The gene editing method according to claim 1, characterized in that when the vector knocks each of the exogenous DNA fragments into the corresponding double-stranded DNA incision, the delivery method of the vector to deliver the exogenous DNA fragment is electroporation, virus or LNP cell. The gene editing method according to claim 13 is characterized in that an inhibitor is added to the delivery system when the exogenous DNA fragment is inserted into the double-stranded DNA incision through the vector. The gene editing method according to claim 14, characterized in that the inhibitor is one or more of a DNA-PK inhibitor, RS-1 and L755507. The gene editing method according to claim 15, characterized in that the inhibitor is a DNA-PK inhibitor including one or both of AZD7648 and M3814. An engineered cell obtained by the gene knock-in method according to any one of claims 1 to 16. Use of the engineered cells according to claim 17 in the preparation of drugs for treating and/or preventing tumors and cancers.