Sgrna highly targeting human HLA-a gene, composition thereof and use thereof

Abstract

A sgRNA. The nucleotide sequence of the sgRNA is as shown in one of SEQ ID NOs: 1-21. The sgRNA can efficiently knock out or knock down a human HLA-A gene, and almost completely covers sgRNAs of HLA-A genotypes of the Chinese population. The sgRNA has high targeting property, has no significant effect on the expression of HLA-B, HLA-C and HLA-II molecules after knocking out or knocking down HLA-A genes in human hematopoietic cells, has low cytotoxicity, exhibits clear advantages in gene therapy, and thus has great clinical application prospects in the field of cell therapy.

Description

sgRNAs that highly target the human HLA-A gene, their compositions and applications

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202410772696.3, filed no earlier than June 17, 2024, entitled "sgRNA highly targeting the human HLA-A gene, its composition and application", the entire contents of which are incorporated herein by reference.

[0003] This application includes a sequence list, which was submitted electronically in .XML format on June 17, 2024. The sequence list contained in the .XML file is part of the specification and is hereby incorporated herein by reference in its entirety. Technical Field

[0004] This disclosure relates to the field of biotechnology, and in particular, to an sgRNA that highly targets the human HLA-A gene, compositions thereof, and applications. Background Technology

[0005] Immune rejection is primarily mediated by the recognition of major histocompatibility complex (MHC) molecules on the surface of non-donor cells by recipient immune cells. Human MHC molecules are encoded by the human leukocyte antigen (HLA) gene, and classic HLA molecules, such as HLA-A, B, C, and DR, are the main molecular targets of allogeneic transplant rejection. Immune rejection is a significant cause of failure for many treatments. For example, hematopoietic stem cell (HSC) transplantation is an important treatment for various malignant hematological diseases and immune dysfunctions; successful HSC transplantation first requires overcoming the transplantation immune barrier (immune rejection).

[0006] Knocking down or eliminating HLA-A can reduce transplant rejection, significantly improve HLA matching success rates, and expand the application of clinical-grade allogeneic grafts. Previous studies have used zinc finger nucleases (ZFNs) to knock out the HLA-A gene in umbilical cord blood-derived hematopoietic cells, improving the matching rate of allogeneic transplantation while maintaining the in vivo engraftment and differentiation capabilities of hematopoietic cells. However, ZFN editing of the HLA-A gene has many drawbacks and limitations. For example, 1) ZFN technology has a low efficiency in knocking out the HLA-A gene, only about 10%, resulting in limited clinical application prospects. 2) ZFN gene editing is prone to off-target effects and produces high cytotoxicity. ZFN DNA cleavage requires dimerization of two Fok I cleaving regions and at least one recognition unit to bind to DNA. Although the DNA recognition domain has strong specific recognition capabilities, the ZFN cleavage process does not entirely depend on the formation of homodimers. Therefore, once heterodimers are formed, off-target effects are likely to occur, potentially leading to DNA mismatches and sequence alterations, resulting in strong cytotoxicity. When these adverse effects accumulate excessively, exceeding the capacity of the cell's repair mechanisms, apoptosis (cell death) can occur. 3) ZFN methods are limited by existing research techniques in the biological field, making the precision and consequences of intracellular manipulation difficult to predict. If ZFN induces mutations in related genes, it could lead to a series of unforeseen consequences, potentially even causing cancer in human-related applications. Currently, ZFN technology can only be used in vitro, processing cells extracted from the human body before reinfusing them into the patient. Directly introducing ZFN elements into the patient for gene editing carries significant potential risks and is inefficient. These limitations make ZFN operations cumbersome and difficult to widely apply. Therefore, there is currently no efficient method for knocking down or eliminating, for example, the HLA-A gene in artificial blood cells; addressing these issues is an urgent priority. Summary of the Invention

[0007] Technical problems to be solved:

[0008] One aspect of the present invention addresses the problem of the lack of efficient means for knocking down and / or knocking out the human HLA-A gene in the prior art by providing a highly targeted sgRNA for the human HLA-A gene, a composition thereof, and its application.

[0009] Specifically, the inventors have creatively designed a small guide RNA (sgRNA) based on the CRISPR gene editing system, which, when applied alone or in combination to the CRISPR gene editing system, can efficiently knock down and / or eliminate the human HLA-A gene, thereby solving the problems in the prior art.

[0010] Technical solution:

[0011] An sgRNA, the nucleotide sequence of which is shown in one of SEQ ID No. 1-21.

[0012] In the embodiments of this disclosure, the human HLA-A gene is recognized, knocked down, and / or eliminated in cells using the aforementioned sgRNA via a CRISPR gene editing system. The efficiency achieved is over 20% in the 293T cell line, and reaches a maximum efficiency of 97% in umbilical cord blood-derived hematopoietic cells, far superior to existing methods. Among these, the sgRNAs HLA-A-ex2-g13, HLA-A-ex3-g167, and HLA-A-ex2-g452 exhibit the highest efficiency. Therefore, preferably, in some embodiments of this disclosure, the nucleotide sequence of the sgRNA is as shown in SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No. 3.

[0013] In some embodiments of this disclosure, the nucleotide sequences shown above, such as SEQ ID No. 1-21, may be reasonably modified. Such modifications include, but are not limited to, substitution, insertion, deletion, or exchange or replacement of one or more nucleotides for improvement purposes, as well as modifications to the nucleotide sequence using other groups.

[0014] Another aspect of this disclosure is to provide a composition of sgRNA comprising one or more sgRNAs selected from the nucleotide sequences shown in SEQ ID No. 1-21.

[0015] Preferably, in some embodiments of this disclosure, the composition comprises one or more sgRNAs selected from the nucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

[0016] In some embodiments of this disclosure, the nucleotide sequence in the above composition may be modified. Such modifications include, but are not limited to, substitution, insertion, deletion, or exchange or replacement of one or more nucleotides for improvement purposes, as well as modifications to the nucleotide sequence using other groups.

[0017] Another aspect of this disclosure is the use of the above-described sgRNA or the above-described composition in the preparation of products for knocking out or knocking down the human HLA-A gene. The knockout or knockdown of the human HLA-A gene can be performed in vivo or in an in vitro culture environment.

[0018] Another aspect of this disclosure is the use of the above-described sgRNA or the above-described composition in the preparation of a medicament for preventing or reducing immune rejection.

[0019] Another aspect of this disclosure is to provide a recombinant vector containing the above-described nucleotide sequence. In some other embodiments of this disclosure, the recombinant vector may also simultaneously load a nucleotide sequence encoding a Cas protein.

[0020] Another aspect of this disclosure is to provide a kit comprising the above-described sgRNA, the above-described composition, or the above-described recombinant vector. In other embodiments of this disclosure, the kit further comprises a Cas protein or an expression vector for the Cas protein.

[0021] Another aspect of this disclosure is to provide a pharmaceutical composition comprising the above-described sgRNA, the above-described composition, or the above-described recombinant vector, and a pharmaceutically acceptable vector.

[0022] Another aspect of this disclosure is to provide a method for improving the knockout or knockdown efficiency of the human HLA-A gene in cells, comprising:

[0023] Step 1) Transform the above-mentioned sgRNA, the above-mentioned composition, or the above-mentioned recombinant vector into the cells;

[0024] Step 2) Transfer the Cas protein or its expression vector into the cell;

[0025] Step 3) describes cell growth.

[0026] In some embodiments of this disclosure, the cells described above can be grown in vivo or in an in vitro culture environment. Beneficial effects:

[0027] The sgRNA disclosed herein can efficiently knock out or knock down the human HLA-A gene, and almost completely covers the sgRNA of the HLA-A genotype in the Chinese population. The sgRNA disclosed herein exhibits high targeting specificity; knocking out or knocking down the HLA-A gene in human hematopoietic cells has no significant impact on the expression of HLA-B, HLA-C, and HLA-II molecules. It also exhibits low cytotoxicity, demonstrating significant advantages in gene therapy and great clinical application prospects in the field of cell therapy. Attached Figure Description

[0028] Figure 1 shows the results of flow cytometry detection of HLA-A expression in 293T tool cells in this embodiment of the present disclosure. The results show that HLA-A protein is highly expressed on the surface of 293T cells and is significantly separated from the negative group. This result indicates that 293T cells can be used as tool cells for HLA-A sgRNA library screening.

[0029] Figure 2 is a flow cytometry result of the virus-infected group after Puro drug screening in this embodiment of the present disclosure. The figure shows the HLA-A low expression cell population. The overall HLA-A expression of cells shifts to the left, and the proportion of the HLA-A low expression population increases. The flow cytometry sorted out 10% of cells with low HLA-A expression, 70% with intermediate expression, and 10% with high expression.

[0030] Figure 3 shows the results of the enrichment of sgRNA in the HLA-A low expression group compared with the unsorted group in the embodiments of this disclosure, thereby screening out 24 effective sgRNAs;

[0031] Figure 4 shows the knockout effect of 21 sgRNAs screened by transient transfection in the 293T cell line in this embodiment of the present disclosure. The figure shows the proportion of HLA-A low expression cells after each sgRNA knockout. The HLA-A knockout cell proportion of the HLA-A-ex2-g13 group with the best knockout effect can reach 60%. In addition, the HLA-A gene site targeted by each sgRNA is marked in the figure, where P is Promoter, Ex is exon, and Int is Intron.

[0032] Figure 5 shows the knockout sites of HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) in the human HLA-A gene in the embodiments of this disclosure;

[0033] Figure 6 shows the results of further testing the knockout effect of the three sgRNAs with the best HLA-A protein knockout effect selected from the 293T cell line in this embodiment of the present disclosure: HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) on hematopoietic cells derived from umbilical cord blood. The flow cytometry plot is CD34. + CD34 + CD90 + and CD34 + CD90 - Cellular gatening strategies.

[0034] Figures 7 and 8 show the knockout effects of HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) on hematopoietic cells derived from umbilical cord blood in the embodiments of this disclosure. Figure 7 shows that all three sgRNAs can effectively knock out CD34. + CD34+ CD90 + and CD34 + CD90 - Cell surface HLA-A protein, 3 sgRNAs knocked out CD34 + CD90 + The cell proportions were all above 94%, and the HLA-A-ex2-g13 (SEQ ID No. 1) group CD34 + CD90 + The proportion of HLA-A knockout cells can reach 97%. Figure 8 shows the statistical analysis, which shows that the proportion of HLA-A positive cells decreased significantly after knocking out 3 sgRNAs.

[0035] Figures 9 and 10 are specific results from embodiments of this disclosure. Figure 9 shows that, compared to the control group, CD34 was significantly reduced after knocking out HLA-A with the three sgRNAs. + CD90 + No significant difference was observed in HLA-ABC expression on the cell surface, indicating that the three sgRNAs targeting and knocking out HLA-A had significant specificity. Knocking out HLA-A with these three sgRNAs did not affect CD34. + CD34 + CD90 + and CD34 + CD90 - The expression of HLA-B and HLA-C molecules on the cell surface was analyzed. Figure 10 shows the statistical analysis. After knocking out the three sgRNAs, the proportion of HLA-ABC positive cells did not change significantly.

[0036] Figures 11 and 12 show the specific results in the embodiments of this disclosure. Figure 11 shows that, compared with the control group, there was no significant difference in the expression of HLA-DR molecules on the surface of CD34+CD90+ cells after knocking out HLA-A with the three sgRNAs. Figure 12 shows the statistical analysis, indicating that the proportion of HLA-DR positive cells did not change significantly after knocking out the three sgRNAs. This demonstrates that the three sgRNAs have significant specificity in targeting and knocking out HLA-A, and that knocking out HLA-A with these three sgRNAs does not affect the expression of HLA-II molecules on the cell surface of CD34+, CD34+CD90+, and CD34+CD90- cells.

[0037] Sequence description.

[0038] sequence list Detailed Implementation

[0039] This invention discloses an sgRNA and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0040] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" refers to two or more. The terms "such as," "for example," etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0041] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0042] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0043] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's *GENES*, Twelfth Edition, by Jocelyn E. Krebs, Elliott S. Goldstein, and Stephen T. Kilpatrick, published by Jones & Bartlett Learning. Definitions of common biochemistry terms can be found in Lehninger's *Principles of Biochemistry*, Eighth Edition, by David L. Nelson and Michael M. Cox, published by WH Freeman. Definitions of common cell biology terms can be found in *Molecular Biology of the Cell*, Sixth Edition, by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, and Peter Walter, published by Garland Science. Definitions of common genetics terms can be found in *Genetics: Analysis of Genes and Genomes*, Eighth Edition, by Daniel L. Hartl and Maryellen Ruvolo, published by Jones & Bartlett Learning.

[0044] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.

[0045] definition:

[0046] The term "knockout" in this disclosure describes the process of completely or partially inactivating a specific gene in an organism using gene editing techniques. Knockout techniques typically involve using gene editing tools, such as the CRISPR-Cas9 system, to precisely locate and cut the DNA sequence of a target gene. After the DNA sequence is cut, cells usually attempt to repair the broken DNA through mechanisms such as non-homologous end joining (NHEJ) or homologous recombination (HDR). However, these repair processes may result in the insertion, deletion, or substitution of gene sequences, causing the target gene to lose its original function or expression level.

[0047] The term "knockdown" in this disclosure refers to reducing the expression level of a gene in an organism through specific technical means, rather than completely eliminating the gene's function, such as through interference from antisense RNA, ribonucleases, or the CRISPR-Cas system (CRISPRi). The main difference between knockdown and knockout lies in the degree: knockout completely or partially eliminates gene function, while knockdown only reduces its expression level. Therefore, knockdown allows researchers to study the effects of reduced gene expression, rather than the effects of complete gene deletion.

[0048] The term "HLA-A" in this disclosure is an abbreviation for Human Leukocyte Antigen A, also known as an MHC class I antigen. It is a glycoprotein expressed on the surface of almost all cells in the human body and is part of the major histocompatibility complex (MHC). The heavy chain of the HLA-A molecule is approximately 45 kDa, and its gene contains eight exons. Exon 1 encodes a leader peptide, exons 2 and 3 encode α1 and α2 domains (both binding peptides), exon 4 encodes an α3 domain, exon 5 encodes a transmembrane region, and exons 6 and 7 encode a cytoplasmic tail. In particular, polymorphisms within exons 2 and 3 are associated with peptide-binding specificity for each HLA-A molecule, and typing of these polymorphisms is commonly used for matching in bone marrow and kidney transplants.

[0049] CRISPR-Cas system:

[0050] The CRISPR-Cas system is widely found on the chromosomes of bacteria and archaea, and is related to their immunity, used to defend against foreign genetic material and acquire resistance to bacteriophages. The CRISPR-Cas system is the third generation of gene editing technology following ZFN and TALENs. The CRISPR-Cas system can be divided into three main types: Type I, Type II, and Type III. CRISPR Type I was the first CRISPR system discovered and studied, and is mainly found in most bacteria and archaea. This system consists of multiple Cas proteins and multiple CRISPR RNAs (crRNAs), forming a complex Cas protein complex. This complex recognizes and cuts the target fragment through complementary pairing of crRNA with foreign DNA. A key characteristic of the Type I system is the presence of multiple Cas proteins, among which the Cas3 protein has RNA-dependent DNA nuclease activity and is responsible for cutting the target DNA. The CRISPR Type II system, also known as the CRISPR / Cas9 system, is currently the most widely used gene editing tool. The CRISPR / Cas9 system requires only the Cas9 protein and crRNA to form a complex with trans-activating CRISPR-derived RNA (tracrRNA) to recognize and cleave target DNA fragments. The Cas9 protein possesses two nuclease activities, HNH and RuvC, which can cleave the two strands of the target DNA, respectively. The CRISPR Type III system has a similar structure and function to the Type I system, but with more Cas proteins and a more complex mechanism. Similar to the Type I system, the Type III system also requires multiple Cas proteins and crRNA to form a complex, recognizing and cleaving target fragments through complementary pairing with exogenous DNA. However, the cleavage mechanism of the Type III system differs from that of the Type I system; it relies on the RNase activity of the Cas proteins to cut the target DNA. In embodiments of this disclosure, the sgRNA is used in the Type II CRISPR-Cas system.

[0051] Cas protein:

[0052] In embodiments of this disclosure, the Cas protein used in the CRISPR-Cas system is primarily Cas9. Cas9 is a nuclease that, guided by sgRNA (small guide RNA), can precisely recognize and cleave DNA sequences. In some embodiments of this disclosure, the Cas9 protein can be isolated or loaded onto a vector for intracellular expression. In addition to Cas9, other accessory proteins may be included in other embodiments of this disclosure, including but not limited to Cas1, Cas2, etc. They may play an auxiliary role in the DNA cleavage process or participate in other aspects of the CRISPR system, such as DNA acquisition and processing.

[0053] In embodiments of this disclosure, the Cas protein may be modified. Such modifications may include mutations, insertions, or deletions of amino acid residues, codon optimization, and other chemical modifications. The purpose may be to alter the activity or specificity of the Cas protein without changing its primary function. For example, US Patent 20180100148A1 describes mutations in the nucleic acid and protein of Streptococcus pyogenes Cas9 to reduce off-target effects of the system.

[0054] sgRNA and its combinations:

[0055] In the embodiments of this disclosure, the nucleotide sequences of the sgRNAs shown in SEQ ID No. 1-21 are derived from an internally constructed (undisclosed) sgRNA library at the Institute of Hematology, Chinese Academy of Medical Sciences. We designed a guide RNA (gRNA) database based on HLA-A gene information (HLA-A major histocompatibility complex, class I, A [Homo sapiens (human)], Gene ID: 3105) provided by NCBI. This library contains 520 sgRNAs, including 50 negative controls. The inventors obtained the sgRNAs and their combinations after screening the library. Knowing the nucleotide sequences, they can be obtained using suitable methods in the prior art, including but not limited to chemical synthesis, polymerase chain reaction (PCR), etc.

[0056] In gene editing, the aforementioned sgRNAs can be used alone, achieving editing efficiencies of approximately 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% in some embodiments of this disclosure. They can also be used in combination or with the addition of other sgRNAs, achieving editing efficiencies of approximately 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% in other embodiments of this disclosure. In some embodiments of this disclosure, the sgRNAs can be loaded into the same container or into different containers before being transfected into cells.

[0057] The nucleotide sequence of the above-mentioned sgRNA can be modified. Such modifications include, but are not limited to, substitution, insertion, deletion, or exchange / replacement of one or more nucleotides for improvement purposes, as well as modifications to the nucleotide sequence using other groups. In some embodiments of this disclosure, the mutated sgRNA nucleotide sequence may have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ No. 1-21.

[0058] Vector or recombinant vector:

[0059] In embodiments of this disclosure, the provided vector, or recombinant vector, contains at least one nucleotide sequence of the sgRNA. The purpose is to transfer nucleic acids to target cells for gene editing. In some embodiments of this disclosure, the vector or recombinant vector also contains a nucleotide sequence encoding a Cas protein, operably linked to a suitable promoter.

[0060] In some embodiments of this disclosure, the vector or recombinant vector may be encapsulated into a virus or virus-like particle for transfer to target cells. Examples of the vector or recombinant vector include, but are not limited to, plasmid vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or retroviral vectors. In some embodiments of this disclosure, the vector may be a linear vector or a circular vector. It may be a non-viral vector such as a plasmid, a viral vector, or a vector utilizing transposons. The vector may contain regulatory sequences such as promoters and terminators, as well as marker sequences such as drug resistance genes and reporter genes.

[0061] Cells used:

[0062] In embodiments of this disclosure, gene editing using the sgRNA or a composition of sgRNA to knock out or down the human HLA-A gene can be utilized on any cell containing human HLA-A. For example, in some embodiments of this disclosure, the cell is a human cell, including but not limited to embryonic stem cells, induced pluripotent stem cells, germ cells, fibroblasts, oligodendrocytes, glial cells, hematopoietic stem cells / hematopoietic progenitor cells, neuronal cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, retinal cells, cancer cells, T cells, B cells, NK cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autologous transplanted expanded cardiomyocytes, adipocytes, differentiated totipotent cells, and multipotent cells. Cells can include pluripotent cells, hematopoietic stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow-derived cells, cardiomyocytes, osteoblasts, fetal cells, undifferentiated cells, pluripotent cells, monopotent cells, monocytes, cardiac myoblasts, osteomyoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, or postnatal stem cells. In some embodiments of this disclosure, the cells may also be cells grown in other organisms but containing human HLA-A.

[0063] In some embodiments of this disclosure, the gene editing can occur in vivo or in vitro. Elements of the CRISPR-Cas system described in this disclosure can be transferred into the cells using any suitable method. Examples include, but are not limited to, transfection, viral infection, electroporation of RNPs, etc.

[0064] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0065] Example:

[0066] The sgRNA library used in this embodiment was internally constructed by the Institute of Hematology, Chinese Academy of Medical Sciences (not publicly disclosed). We designed a guide RNA (gRNA) database based on HLA-A gene information (HLA-A major histocompatibility complex, class I, A [Homo sapiens (human)], Gene ID: 3105) provided by NCBI. This library contains 520 sgRNAs, including 50 negative controls.

[0067] Example 1: HLA-AsgRNA library screening

[0068] 1. First, the expression level of HLA-A on the surface of 293T cells was detected. The 293T cells were cultured in DMEM complete medium: DMEM + 10% FBS + 1% penicillin / streptomycin.

[0069] 1) Both the experimental group and the control group were tested at 2×10⁻⁶. 5 Cells, experimental group cells were labeled with HLA-A2 APC-Cy7 antibody and incubated at 4°C for 30 minutes.

[0070] 2) Add 1 ml of PBE buffer to wash the antibody, centrifuge at 1500 rpm for 5 min and discard the supernatant. Resuspend the cells in 300 μL of PBE + 2% FBS. Before testing, dilute with DAPI at a ratio of 1:1000.

[0071] 3) Flow cytometry results showed that 293T cells exhibited high expression of HLA-A protein on their surface and were significantly separated from the negative control group. This result indicates that 293T cells can be used as tool cells for HLA-AsgRNA library screening (results are shown in Figure 1).

[0072] 2. Packaging lentivirus from HLA-AsgRNA plasmid library. The virus was packaged in 10cm culture dishes. The virus packaging system is as follows (Table 1):

[0073] Table 1

[0074] 1) Replace the DMEM complete medium with 6 ml of Optim medium and place it in an incubator.

[0075] 2) Prepare the transfection system: First, add 1 ml of Optim medium to the EP tube, and then add the vortexed PAX2, VSVG, and sgRNA library plasmids according to the above system.

[0076] 3) Vortex, short centrifugal force.

[0077] 4) Add PEI according to the specified amount and mix thoroughly by blowing.

[0078] 5) Let stand for 20 minutes.

[0079] 6) Add the plasmid system to the dish and shake well.

[0080] 7) After 8 hours, switch to DMEM complete culture medium.

[0081] 8) Collect the viral supernatant after 48 hours and place it at 4℃.

[0082] 3. Measure the virus titer.

[0083] 1) One day in advance, seed 293T cells into 48-well plates, 6 × 10⁶ cells per well. 4 Cells, 300 μL DMEM complete medium.

[0084] 2) Centrifuge the collected viral supernatant at 2000g for 5 minutes. Filter the viral supernatant using a 0.45um filter.

[0085] 3) Prepare the virus infection system according to Table 2, with 500 μL of infection system per well, and prepare 6 virus gradients, with two replicates for each gradient. In addition, prepare 6 control wells without virus.

[0086] Table 2

[0087] 4) Aspirate the culture medium from the culture plate and add it to the virus infection system.

[0088] 5) 48 hours after infection, add 2ug / ml of puromycin to one replicate well of each virus gradient group and the control group, and use the other well as the control.

[0089] 6) After 48 hours of puromycin screening, all cells in the control group should be observed to be dead. Record the number of viable cells in the puromycin group and the group without puromycin for each viral gradient.

[0090] 7) Calculate the viral gradient according to the formula:

[0091] (60000 * (number of viable cells in the puromycin group / number of cells in the non-purulent group)) / virus volume (ml) = virus titer TU / ml

[0092] 4. Infect Cas9-stable 293T cell lines with viral supernatant.

[0093] 1) One day in advance, seed cells in 10cm dishes, calculating cell count and virus volume to ensure a virus infection rate of less than 30%. Infection system: x ml virus supernatant + (10-x) ml DMEM complete medium + 10 μL polybrene. Perform 3 replicates and 1 control group without virus.

[0094] 2) 2 μg / ml puromycin was added for screening 48 hours after viral infection. HLA-A knockout was detected by flow cytometry on day 17 of screening. An increase in the proportion of HLA-A-low expression cells was observed in the viral group compared to the control group. Flow cytometry was used to sort out the 10% HLA-A-low expression cell population and the 10% HLA-A-high expression cell population, while retaining the unsorted cell population (results shown in Figure 2).

[0095] 5. Extract the genomes of HLA-A low-expression cell populations and unsorted cell populations, amplify sgRNA fragments using PCR technology, and perform NGS sequencing on the amplified sgRNA fragments.

[0096] 1) Extract the genomes of HLA-A low-expressing cell populations and unsorted cell populations using the TIANGEN DNA extraction kit and measure the DNA concentration in each group.

[0097] 2) The sgRNA fragment was amplified using PCR technology, involving two PCR steps:

[0098] PCR1 (345bp)

[0099] Primers:

[0100] CRISPR_PCR1_F:5'-AGGGCCTATTTCCCATGATT

[0101] CRISPR_PCR1_R:5'-CGGTGCCACTTTTTCAAGTT

[0102] PCR1 system and conditions:

[0103] PCR2 (265bp)

[0104] Primers:

[0105] B5xx_CRISPR_PCR_2F:5'-:NNNNNNNNAGGCTGTTAGAGAGATAA

[0106] B7xx_CRISPR_PCR_2R:5'-NNNNNNNNGCTGTTTCCAGCATAG

[0107] PCR2 system and conditions (8 reactions per sample):

[0108] 6. Purify the PCR products according to the ZYMO RESEARCH DNA purification kit method, and measure the DNA concentration after purification. Send 500 ng of each sample for next-generation sequencing.

[0109] 7. Based on the sequencing results, bioinformatics analysis was performed on the enrichment of sgRNAs in the HLA-A low expression group compared to the unsorted group. A Fold change greater than 1.5 and p < 0.05 were used as criteria, and a total of 24 effective sgRNAs were screened (results shown in Figure 3).

[0110] 8. The China MAP database (http: / / www.mbiobank.com) was consulted. This database contains the total number of HLA gene variant sites (SNPs) and their frequencies in the Chinese population. All HLA-A SNPs (95 mutation sites) provided by the database were compared with the 24 effective sgRNAs selected above, and 3 sgRNAs containing variant sites were excluded. Since the SNP frequency provided by this database is less than one in ten thousand, the 21 selected sgRNAs (excluding variant sites) are considered to cover >99% of the Chinese population. Their sequences are shown in SEQ ID No. 1-21.

[0111] Example 2: The knockout effect of the selected sgRNA was tested on the 293T cell line using transient transfection.

[0112] 1) 293T cells were seeded in 24-well plates 2 × 10⁶ cells per well 24 hours in advance. 5 cell.

[0113] 2) Replace the DMEM complete medium with 0.4 ml of DMEM medium (containing 10% FBS, without penicillin / streptomycin) and place it in an incubator.

[0114] 3) Prepare the transfection system: Add 50ul of Optimum medium to a 1.5ml EP tube, then add 1ug / well of plasmid (0.5ug / well each of sg plasmid and Cas9 plasmid, sg plasmid is prepared using conventional methods), invert and mix well, do not vortex.

[0115] 4) Take another 1.5ml EP tube, add 50ul of Optimum medium, then add 3ug / well of PEI, invert to mix, do not vortex.

[0116] 5) Leave at room temperature for 5 minutes.

[0117] 6) Mix 3) and 4) in one tube, invert to mix well, do not vortex, and let stand for 20 minutes.

[0118] 7) Add the plasmid system to the dish and shake well.

[0119] 8) After 12-16 hours, switch to DMEM complete culture medium.

[0120] 9) Add 2ug / ml puromycin vial sieve 48 hours after transfection.

[0121] 10) The HLA-A knockout effect of each sgRNA was detected 48 hours after puromycin screening.

[0122] 11) Flow cytometry results showed that among the 21 sgRNAs, HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) had better knockout effects, with the HLA-A-ex2-g13 group achieving a HLA-A knockout rate of up to 60% (see Figure 4 for results).

[0123] 12) The knockout sites of HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3) on the human HLA-A gene are shown in Figure 5.

[0124] Example 3: Detection of the knockout effect of screened sgRNAs on umbilical cord blood-derived hematopoietic cells

[0125] 1. Enrichment of umbilical cord blood CD34 + Cells were incubated overnight in hematopoietic cell basal medium. The hematopoietic cell basal medium was SFEMII medium supplemented with the following cytokines (Table 3):

[0126] Table 3

[0127] 2. Three sgRNAs with good knockout effects in 293T cells were selected: HLA-A-ex2-g13 (SEQ ID No. 1), HLA-A-ex3-g167 (SEQ ID No. 2), and HLA-A-ex2-g452 (SEQ ID No. 3). CD34 was knocked out using the electroporation RNP method according to the Lonza P3 Primary Cell 4D-NucleofectorTMX Kit S. + HLA-A protein on the cell surface.

[0128] 1) CD34 + Cell count, 2 × 10⁻⁶ per sample 5 cell.

[0129] 2) Prepare the electroporation system: Add buffer to each tube first, then add 75 pmol spCas9 protein and 150 pmol sgRNA, for a total of 5 μL. Incubate at room temperature for 20-30 min.

[0130] 3) Resuspend the cells in 15 μL of buffer, add RNP (to make a 20 μL system), and pipette the system >10 times. Add liquid, being careful not to add air bubbles.

[0131] 4) Operate according to the instructions of the 4D electro-rotator.

[0132] 5) After electroporation, transfer the cell suspension from the electroporation tank to a 1.5ml centrifuge tube. Centrifuge at 500g for 5 minutes and discard the supernatant.

[0133] 3. Transfer cells to 24-well plates for culture and observation. After 72 hours, CD34 levels were detected by flow cytometry. + CD34 + CD90 + and CD34 + CD90 - Expression of HLA-A, HLA-ABC, and HLA-DR on the cell surface. The antibodies labeled by flow cytometry are shown in Table 4 below.

[0134] Table 4

[0135] 4. Flow cytometry results showed that all three sgRNAs could significantly knock out CD34. + CD34 + CD90 + and CD34 + CD90 - HLA-A protein on cell surface, 3 sgRNAs knocked out CD34 + The cell proportions were all above 94%, and the HLA-A-ex2-g13 group had CD34. + CD90 + The proportion of HLA-A knockout cells reached 97% (Figure 6 shows the flow cytometry gating strategy; results are shown in Figures 7 and 8). Furthermore, the three sgRNAs group CD34... + CD34 + CD90 + and CD34 + CD90 - The proportions of HLA-ABC and HLA-DR positive groups on cells did not change significantly. After statistical analysis, knocking out HLA-A with the three sgRNAs had no significant effect on the expression of HLA-B and HLA-C in HLA-I molecules in the three cell populations (see Figures 9 and 10) or HLA-II molecules (see Figures 11 and 12). This means that the sgRNAs we used have high targeting specificity.

[0136] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An sgRNA, characterized in that, The nucleotide sequence of the sgRNA is shown in one of SEQ ID No. 1-21.

2. The sgRNA according to claim 1, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No.

3.

3. The sgRNA according to claims 1-2, characterized in that, The nucleotide sequence is modified.

4. A composition of sgRNA, characterized in that, The composition comprises one or more sgRNAs selected from the nucleotide sequences shown in SEQ ID No. 1-21.

5. The composition according to claim 4, characterized in that, The composition comprises one or more sgRNAs selected from the nucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No.

3.

6. Or the composition according to claims 4-5, characterized in that, The nucleotide sequence is modified.

7. The use of the sgRNA as described in claims 1 to 3 or the composition as described in claims 4 to 6 in the preparation of products for knocking out or knocking down the human HLA-A gene.

8. The use of the sgRNA as described in claims 1 to 3 or the composition as described in claims 4 to 6 in the preparation of a medicament for preventing or reducing immune rejection.

9. A recombinant vector, characterized in that, The recombinant vector contains the nucleotide sequence as described in any one of claims 1 to 6.

10. The recombinant vector according to claim 9, characterized in that, The recombinant vector also contains a nucleotide sequence encoding the Cas protein.

11. A reagent kit, characterized in that, The kit includes the sgRNA as described in claims 1-3, the composition as described in claims 4-6, or the recombinant vector as described in claims 9-10.

12. The kit according to claim 11, characterized in that, The kit also includes the Cas protein or an expression vector for the Cas protein.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the sgRNA as described in claims 1-3, the composition as described in claims 4-6, or the recombinant vector as described in claims 9-10, as well as pharmaceutically acceptable vectors.

14. A method for improving the knockout or knockdown efficiency of the human HLA-A gene in cells, characterized in that, include: Step 1) Transform the cells with the sgRNA as described in claims 1-3, the composition as described in claims 4-6, or the recombinant vector as described in claims 9-10; Step 2) Transfer the Cas protein or its expression vector into the cell; Step 3) describes cell growth.

15. The method according to claim 14, characterized in that, The cells are grown in vivo or in an in vitro culture environment.

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