Cancer is a genetic disease accompanied by the accumulation of a variety of mutations (1). Tumor immunotherapy has become one of the most promising therapeutic strategies in tumor treatment compared with the high recurrence rate of metastasis, drug resistance and poor prognosis of traditional therapies (2). Tumor cells actively evade immune detection by activating associated negative regulatory pathways (also known as checkpoints), thus inhibiting the immune response of the human body. Among the numerous checkpoints, cytotoxic T lymphocyte protein 4 (CTLA4) and programmed cell death protein 1 (PD-1) are by far received much attention. CTLA4 controls T cell activation by competing with the costimulatory molecule CD28 to bind to the shared ligands CD80 and CD86 (3). The binding of PD-1 on the surface of T cells to Programmed death ligand 1(PD-L1) on tumor cells is a major obstacle in the cancer immune cycle, inducing T cell apoptosis and inhibiting the activation and proliferation of T cells (4). Fortunately, genetic modification of tumor immunity can be achieved through the CRISPR/Cas9 system. The CRISPR/Cas9 system, derived from the acquired immune defense mechanism of bacteria, enables accurate editing of specific regions of the genome by simple and rapid Watson Crick base pairing between sgRNA and target genes (5, 6). Currently, the CRISPR/Cas9 system has derived many variants such as nickase Cas9 (nCas9), nuclease-deactivated Cas9 (dCas9) and so on, which have been widely used in a variety of cell types and organisms to achieve diversity and ease of use (7, 8). In this review, we summarize CRISPR/Cas9 technology centering on gene editing, gene screening and clinical application of tumor immunity PD-1/PD-L1, as well as its strong potential and existing problems in the field of future tumor therapy.

Tumors are characterized by highly heterogeneous molecular characteristics and carry a wide range of gene mutations (9). This may be the main cause of tumor drug resistance and recurrence (10). Gene editing technology is in urgent need of decoding the genetic code at a deeper level and implementing precision medicine. Genome editing can modify the human genome and change biogenesis, which has the potential of targeted therapy to cure diseases (11). The CRISPR/Cas9 system is a gene editing technology guided by the principle of RNA-DNA complementary pairing (12). CRISPR/Cas9 technology is used to repair mutations, knock in or knock out specific genes, and modify genes to explore the mechanisms of tumorigenesis, development, and metastasis for precision therapy. The CRISPR sequence is responsible for the production of crRNA and tracrRNA ( Figure 1 ). The two can be synthesized into a single-stranded guide RNA (sgRNA) (6, 13), which guides Cas9 nuclease to bind to the complementary target gene and cut near the protospacer adjacent motif (PAM) region to introduce DNA double strand breaks(DSBs) (5, 14). At DSBs, Non-homologous end-joining (NHEJ) repair pathway is activated to randomly insert or delete several bases, and efficiently produce INDEL mutations (15). Alternatively, Homologous directed repair (HDR) can occur under a DNA repair template with high homology (16). Compared with Zinc finger endonuclease (ZFN) (17) and Transcription activator-like effector nuclease (TALEN) (18) technologies, the CRISPR/Cas9 system abandons the traditional chimeric nuclease protein domain-DNA recognition design concept and avoids the multiple fusion process in multitarget gene editing (19–21). Therefore, it has the advantages of high specificity, strong operability and convenient systematic analysis, making it the preferred tool for gene editing in eukaryotes. CRISPR/Cas9 gene editing technology has been widely used in the construction of animal tumor models, the study of drug resistance regulation mechanisms, epigenetic control and tumor immunotherapy (22–24). In particular, CRISPR/Cas9 also shows great potential in exploring tumor immune mechanisms, biomarker screening, and clinical trials and therapies.

The occurrence and development of tumours are closely related to the infiltration of immune cells, immune modification and immune escape in the tumour microenvironment (1, 25, 26). Tumour immunity promotes tumour progression by changing tumour biological characteristics (27), screening tumour cells adapted to the microenvironment for survival (28) or establishing a suitable tumour microenvironment (29), and so on. Among them, immune checkpoints such as PD-1 and CTLA4 play an important role. Under normal physiological conditions, the binding of PD-1 and PD-L1 can down-regulate the activity of T cells and prevent additional damage of cytotoxic effector molecule and autoimmunity, so it is called immune checkpoint (30) A Study confirmed that tumor cells often express negative costimulatory signals such as PD-L1, leading to the failure activation of T cell activation. At the same time, tumor-infiltrating lymphocytes (TILs) usually express elevated levels of PD-1 due to chronic stimulation of tumor antigens (31). PD-1/PD-L1 blockade enhances T cell response through multiple underlying mechanisms, altering the outlook for cancer treatment. For example, PD-1/PD-L1 blockade may depend on proliferation of "precursors of exhausted" T (TPEX) cells rather than reversal of T cell depletion procedures (32). It has been found that PD-L1 blockade in conjunction with Alarmin HMGN1 peptide seems to activate anti-tumor TPEX cells and promote their amplification, but does not deplete T cells (33). In addition, PD-1 posttranslational modifications such as glycosylation maintain its stability and membrane expression, and mediate immunosuppressive function. Antibodies targeting glycosylated PD-1 may recognize the heavy glycan moieties of PD-1 and have a higher affinity (34).

Tumor immunity is regulated by many factors and ways. For example, iron poisoning can induce immunogenic apoptosis or necroptosis in cancer cells, which activates antitumor immunity (35). Autophagy regulation inhibits the response through selective degradation of MHC class I molecules, reduces the activity of the STING1 pathway, and promotes tumor immune escape (36). In recent years, CRISPR/Cas9 technology has received increasing attention in tumor immune regulation. CRISPR/Cas9 plays a role in the IFN signaling pathway, ERK/MAPK signaling pathway, Wnt/β-catenin signaling pathway, PI3K/AKT signaling pathway and other mechanisms in chronic inflammation and tumor immune resistance. For example, Wang et al. found that knockout of SHP2 using CRISPR/Cas9 gene editing inhibited SHP2 activity and enhanced tumor-intrinsic IFNγ signaling, resulting in increased chemoattractive cytokine release and cytotoxic T cell recruitment, as well as increased tumor cell surface Increased expression of MHC class I and PD-L1. In addition, SHP2 inhibition reduced the differentiation and suppressive function of immunosuppressive myeloid cells in the tumor microenvironment (37). The study by Yang et al. found that successful nuclear localization of CRISPR/Cas9 ensured efficient destruction of PD-L1 and PTPN2. Inhibition of PTPN2 can alleviate the inhibition of the JAK/STAT pathway and promote tumor susceptibility to CD8+ T cells dependent on IFN-γ, thereby further amplifying the adaptive immune response (38). Therefore, the combination of CRISPR/Cas9 technology with tumor immunotherapy has great potential for development. Immune checkpoint block is one of the most valuable individualized tumor treatment options. For example, the anti-PD-1/PD-L1 antibodies, nivolumab, pembrolizumab, and atezolizumab have shown significant advantages in melanoma, non-small-cell lung cancer (NSCLC), and urothelial carcinoma, and have been approved by the Food and Drug Administration (39–41). However, there remains inflammatory side effects (42, 43), and the overall survival rate is not significantly improved. In addition, CPISPR/Cas9 technology provides a multifunctional and convenient operation mode for the transformation of engineered T cells (44, 45). Genetically engineered T cells have the ability to kill tumor cells as well as significant therapeutic effects. Gene editing technology enables precise genetic modification of T cells, further improving chimeric antigen receptor-engineered T cell immunotherapy. This solves the problem of mass production of therapeutic immune cells and enables the genetic engineering of multiple T cells to meet the clinical needs for the treatment of complex types of cancer.

PD-1/PD-L1 has been identified as a negative immunomodulatory molecule that promotes immune evasion of tumor cells. Most patients treated with commercially targeted PD-1/PD-L1 antibodies have achieved a higher cure rate, prolonged survival and fewer recurrence and metastasis events (46–48). However, there are still some patients who are insensitive to targeted PD-1/PD-L1 antibodies or suffer side effects or drug-related inflammatory adverse events (49). The CRISPR/Cas9 technique maximizes PD-1/PD-L1 deletion at the genomic level, thereby saving the function of lymphocytes in the tumour microenvironment. However, whether it corrects the inactivation of targeted antibodies remains unclear ( Figure 2 ).

The occurrence and development of tumors is a complex pathological process involving the interaction of multiple regulatory molecules and their downstream signaling pathways. CRISPR/Cas9 has enabled further understanding of the regulatory mechanism of immune checkpoint inhibitors (ICIs) and promising biomarkers to help patients stratify and coordinate targeted anti-PD-1/PD-L1 therapy.

The occurrence and progression of tumors are closely related to gene mutations (1). Genome-wide screening can comprehensively and fairly grasp the changes in genes in cells, reveal the genetic basis behind specific phenotypes and assist in the development of new therapies. Advances in whole-genome screening in mammalian systems have been hampered by time, size constraints and inefficient double-allelic mutations in mouse culture (77). In recent years, CRISPR/Cas9 technology has provided genome-scale gRNA libraries in the presence of gRNA-guided Cas9 endonucases to generate mutant cell libraries, which serve as a perfect platform for phenotypic screening (78). At present, Cas9 technology has been used in the laboratory, and various sgRNA libraries constructed have revealed the internal molecular regulatory mechanisms of tumor proliferation and metastasis (79), tumor drug resistance (80, 81), tumor immune escape (82) and other malignant behaviors. Below, we summarize the recent progress of CRISPR/Cas9 technology in screening PD-1/PD-L1 and potential regulatory molecules in immune-related cells and tumor cells.

CRISPR technology was used to conduct genome-wide screening of tumor-infiltrating CD8 T cells in vivo. They reidentified typical immunotherapy targets such as PD-1 and TIM-3, as well as other novel sites, and verified the region of CRISPR in vivo screening of CD8 T cells for tumour infiltration (83). Another study identified Fut8, a gene involved in the core focusing pathway, as a positive regulator of PD-1 expression on the cell surface through genome-wide functional loss screening of the CRISPR-Cas9 system. Inhibition of Fut8 can reduce PD-1 expression on the cell surface, enhance T cell activation, and eradicate tumors more effectively (84). Using genome-wide CRISPR and metabolic inhibitor screening, Wang determined that nicotinamide phosphoribotransferase (NAMPT) is required for T cell activation and demonstrated that NAD+ supplementation significantly enhances anti-PD-1 immunotherapy in a mouse solid tumor model (85). This demonstrates the feasibility of CRISPR/Cas9 screening technology in discovering PD-1-related regulatory factors and provides a more comprehensive strategy for in-depth exploration of the PD-1 regulatory mechanism.

In addition to screening in T cells, CRISPR/Cas9 also showed great strength in the study of PD-L1 expressed in tumor cells. At the transcriptional level, CRISPR/Cas9 screening confirmed that ALK activated STAT3 and eventually induced PD-L1 expression through the effects of the transcription factors IRF4 and BATF3 on PD-L1 gene enhancer regions (86). At the translation level, the overexpression of translation initiation factor eIF5B in lung adenocarcinoma allowed us to bypass the inhibitory upstream open reading frame of PD-L1 mRNA in the integrated stress response (ISR) activated by impaired haem production, leading to enhanced PD-L1 translation (87). CMTM6 prevents PD-L1 from lysosomal-mediated degradation, which colocalizes with PD-L1 in the plasma membrane and circulating endosomes (88). In addition, Aleksandra developed a pro-code/CRISPR screen that identified socs1 as a negative regulator of PD-L1 (89). In the same way, a study identified IRF2 in a CRISPR-based forwards genetic screen that controlled MHC-I Ag presentation and PD-L1 expression in HeLa cells (90). CRISPR/Cas9 screening found that the transcription regulator MLLT6 is required for effective PD-L1 protein expression and cell surface presentation in cancer cells. MLLT6 loss mitigates the inhibition of CD8 cytotoxic T cell-mediated lysis (91). In addition, CRISPR/Cas9 screening also found that editing Asf1a (69), PRMT1 and RIPK1 (92), MAN2A1 (93), IFNGR2 and JAK1 (94) increased the sensitivity of tumors to anti-PD-1 or PD-L1 treatment. Tumor cells disrupt T-cell-mediated immune monitoring by maintaining high levels of PD-L1, while CRISPR/Cas9 screening found that the PD-L1 transcription and level, translation levels and maintenance of its stability and expression of oncogenic factors, thus exploring the related regulatory mechanisms of PD-L1 to provide a potential biologic therapeutic target for disrupting PD-L1-mediated tumor tolerance therapy.

The dominant mechanism of tumor immunity is different in different tumors, which affects the occurrence and progression of tumors in various ways. In this review, we selected several cancers and summarized the role of CRISPR/Cas9 technology-associated PD-1/PD-L1 editing in tumor immunity ( Figure 3 ).

Immunotherapy has become a powerful treatment for many solid and hematological malignancies, including immune checkpoint blocking therapy, immune cell therapy and tumor vaccine therapy (124). Among them, CAR T cell therapy in immune cell therapy has shown great potential in the field of tumor therapy and made unprecedented breakthroughs in clinical practice (98, 125). CAR T therapy is administered by taking T cells from patients and genetically modifying them into chimeric antibody T cells with single-stranded fragment variants from the extracellular antigen recognition domain, the CD3ζ transmembrane domain, and the intracellular T cell activation domain (126). It can specifically recognize tumor cell surface-associated tumor-associated antigen, enhance its defense ability against tumor cells, and stimulate T cell proliferation, cytokine secretion, and lymphocyte recruitment, thereby playing an antitumor role. Fortunately, four generations of evolution have improved the time, difficulty and cost of obtaining CAR T cells, as well as the limitations on the number of patients’ T cells themselves (127). The use of CRISPR/Cas9 gene editing technology opens a new world for the upgrading of fourth-generation CAR T cells ( Figure 4 ). Recently, several clinical trials by Stadtmauer (128), Lu (129), Wang (130), and Simon (131) confirmed the clinical feasibility and safety of CRISPR/Cas9- modified T cells.

First, CRISPR/Cas9 can be simultaneously transferred into multiple gDNAs to edit multiple genes at once, providing new ideas for general-purpose allogeneic CAR T cells. For example, Ren by combining CAR lentivirus delivery and electrical transfer of Cas9 mRNA and gRNA, simultaneously targeted endogenous TCR, B2 M and PD-1 and produced allogeneic CAR T cells lacking TCR, HLA class I molecules and PD1 gene destruction. It shows strong antitumor activity in vitro and in animal models, with reduced allograft T cell rejection, and does not cause graft-versus-host disease (45). At the same time, they developed a universal system for the generation of CAR T cells by rapid multigene editing, adding multiple gRNAs into CAR lentivirus vectors and redirecting T cells with antigen-specific CAR through a single CRISPR protocol (100). Second, CRISPR/Cas9-mediated disruption of immune checkpoint signaling, such as PD-1, enhances the therapeutic efficacy of CAR T cells in the immunosuppressive tumor microenvironment. Initially, three studies were completed in vitro in a preclinical model of human glioblastoma to demonstrate that engineered PD-1-deficient EGFRvIII CAR T cells (132, 133), or CD133-specific CAR T cells (134) showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro and enhanced tumor growth inhibition in a mouse model of glioma in situ. Similarly, the combination of positive stimulation from CAR and negative regulation of PD-1 by Cas9 blocking can induce T cells to be more persistent and invasive in vivo in triple-negative breast cancer TNBC (135). In addition, PD-1-deficient GPC3-CAR T cells significantly increased the phosphorylation of Akt and expression of the antiapoptotic protein Bcl-XL, thus preventing the depletion of PD-1-deficient GPC3-CAR T cells from confronting hepatocellular carcinoma cells expressing natural PD-L1 (112). Nevertheless, altering the proportion of PDCD1-deficient CARS affects T cell metastasis, meaning that a strong loss of PDCD1 may enhance CAR T cells in the short term but ultimately make the edited cells more likely to fail or function impaired. Therefore, further studies on the interaction of tumor load, T cell number and editing frequency in different tumors are needed to ensure that CRISPR/Cas9 plays a positive role (136). Subsequently, the application of Cas9-edited PD-1 in CAR T cells has been progressively implemented in clinical trials such as phase I clinical trials of multiple myeloma with mesothin-positive solid tumors (NCT03545815), multiple myeloma (NCT03399448), esophageal cancer (NCT03081715), metastatic NSCLC (NCT02793856), EBV (Epstein-Barr virus)-positive advanced stage malignancies (NCT03044743), advanced hepatocellular carcinoma (NCT04417764) and engineered TILs/CAR-TILs to treat advanced solid tumors (NCT04842812). Overall, CRISPR technology has brought new vitality to CAR T cells in the field of tumor immunotherapy and has gradually entered the stage of clinical trials.

In summary, CRISPR/Cas9 gene editing technology has great potential in the field of tumor PD-1-related immunotherapy. CRISPR/Cas9 gene editing technology can directly edit PD-1/PD-L1 or indirectly regulate antigen presentation and upstream and downstream signaling pathways to improve a variety of immune cells and the tumor microenvironment, and combat tumor immunosuppression. CRISPR genome-wide screening technology also provides new ideas for studying upstream and downstream regulatory mechanisms of PD-1/PD-L1, screening synergistic targeting molecules and identifying potentially sensitive patients. CRISPR/Cas9 has clinical value in the diagnosis and treatment of leukaemia, multiple myeloma, breast cancer, cervical cancer, ovarian cancer, liver cancer, colorectal cancer, lung cancer and melanoma. CRISPR/Cas9 produces allogeneic general-purpose CAR T cells combined with immune checkpoint knockout, expanding the clinical application of engineered cells.

However, the following problems need to be overcome when transforming from laboratory to clinical application: First, off-target effects: gene mutations caused by off-target cutting are prone to cellular malignancy but lead to cancer. Therefore, it is necessary to design sgRNA sequences to increase the specificity of target site cleavage (13) and develop Cas9 variants with PAM preference changes (137). Alternatively, the function of the P53 gene should be monitored to prevent toxicity caused by P53 mutations (138). Second, the activity and proliferation of edited cells should ideally be unaffected. Currently, gene editing and cell amplification can be performed in vitro and transfused back into patients for treatment combined with adoptive cell therapy. Third, safe and effective in vivo delivery methods were selected. At present, the most conventional delivery system is viruses, but they have the disadvantages of limited delivery scale and long-term accumulation easily leading to untargeted cutting. In addition, electroporation and nucleotide transfection techniques have been further explored in animal models. At present, lipid nanoparticle-mediated nonviral delivery without limitation of immunogenicity is more promising (139). Last, Cas9 is an inherent immunogenicity of foreign proteins. Current studies have shown that Cas9 can eliminate immune dominant epitopes through targeted mutations while retaining its function and specificity (140). In brief, although CRISPR/Cas9 gene editing technology shows great promise for PD-1/PD-L1-related therapies in tumor immunity, it is still in its infancy and requires a more comprehensive and in-depth investigation of its safety and reliability.

ZS provided direction and guidance throughout the preparation of this manuscript. YX wrote and edited the manuscript. CC and YG reviewed and made significant revisions to the manuscript. SH collected and prepared the related papers. All authors read and approved the final manuscript.

This study was supported by The National Natural Science Foundation of China (81972663, 82173055, U2004112), The Excellent Youth Science Project of Henan Natural Science Foundation (212300410074), The Key Scientific Research Project of Henan Higher Education Institutions (20A310024), The Youth Talent Innovation Team Support Program of Zhengzhou University (32320290) and The Provincial and Ministry co-constructed key projects of Henan medical science and technology (SBGJ202102134).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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