key: cord-0017800-e7k5ydtg
authors: Alves, Eric; Taifour, Shahama; Dolcetti, Riccardo; Chee, Jonathan; Nowak, Anna K.; Gaudieri, Silvana; Blancafort, Pilar
title: Reprogramming the anti-tumor immune response via CRISPR genetic and epigenetic editing
date: 2021-04-24
journal: Mol Ther Methods Clin Dev
DOI: 10.1016/j.omtm.2021.04.009
sha: 48bf1e102ffac5a965cdb73a9355045b2a8296d8
doc_id: 17800
cord_uid: e7k5ydtg

Precise clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic and epigenetic manipulation of the immune response has become a promising immunotherapeutic approach toward combating tumorigenesis and tumor progression. CRISPR-based immunologic reprograming in cancer therapy comprises the locus-specific enhancement of host immunity, the improvement of tumor immunogenicity, and the suppression of tumor immunoevasion. To date, the ex vivo re-engineering of immune cells directed to inhibit the expression of immune checkpoints or to express synthetic immune receptors (chimeric antigen receptor therapy) has shown success in some settings, such as in the treatment of melanoma, lymphoma, liver, and lung cancer. However, advancements in nuclease-deactivated CRISPR-associated nuclease-9 (dCas9)-mediated transcriptional activation or repression and Cas13-directed gene suppression present novel avenues for the development of tumor immunotherapies. In this review, the basis for development, mechanism of action, and outcomes from recently published Cas9-based clinical trial (genetic editing) and dCas9/Cas13-based pre-clinical (epigenetic editing) data are discussed. Lastly, we review cancer immunotherapy-specific considerations and barriers surrounding use of these approaches in the clinic.

Precise clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic and epigenetic manipulation of the immune response has become a promising immunotherapeutic approach toward combating tumorigenesis and tumor progression. CRISPR-based immunologic reprograming in cancer therapy comprises the locus-specific enhancement of host immunity, the improvement of tumor immunogenicity, and the suppression of tumor immunoevasion. To date, the ex vivo re-engineering of immune cells directed to inhibit the expression of immune checkpoints or to express synthetic immune receptors (chimeric antigen receptor therapy) has shown success in some settings, such as in the treatment of melanoma, lymphoma, liver, and lung cancer. However, advancements in nuclease-deactivated CRISPR-associated nuclease-9 (dCas9)mediated transcriptional activation or repression and Cas13directed gene suppression present novel avenues for the development of tumor immunotherapies. In this review, the basis for development, mechanism of action, and outcomes from recently published Cas9-based clinical trial (genetic editing) and dCas9/Cas13-based pre-clinical (epigenetic editing) data are discussed. Lastly, we review cancer immunotherapyspecific considerations and barriers surrounding use of these approaches in the clinic.

The human immune response consists of a complex and diverse array of molecular and cellular processes to differentiate between self and non-self, which allows it to defend and protect the host from pathogen infection, cellular damage, or neoplastic transformation. In the context of cancer, the constant selective pressure exerted by the host immune response often results in the selection of tumor variants capable of immune evasion that enable tumor cells to survive. Broadly, these tumor evasion mechanisms can involve (1) the accumulation of suppressive cells, such as CD4 + CD25 + FoxP3 + regulatory T cells (Tregs), in the cellular microenvironment; 1,2 (2) down-modulation of the antigen processing and presentation pathways within the cancer cell; [3] [4] [5] (3) shedding of stress, damage, or transformation markers at the cancer cell surface, including the six UL16 binding proteins (ULBP1-ULBP6) and major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA) and B (MICB); [6] [7] [8] (4) secretion of immunosuppressive cytokines, particularly transforming growth factor b (TGF-b), vascular endothelial growth factor (VEGF), and interleukin-10 (IL-10), into the surrounding microenvironment; 9-12 and (5) upregulation of immune checkpoint ligands, especially those pertaining to the programmed cell death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) pathways. [13] [14] [15] [16] [17] Although the last few decades have brought an improved understanding of the underlying molecular mechanisms of these immune evasion strategies, the development of safe and broadly effective immunotherapies that overcome these barriers and are applicable to multiple cancer types remains a formidable task. Accordingly, considerable research focus has been directed toward harnessing the high specificity of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic and epigenetic editing as emerging precision therapeutics to counter the aforementioned immune evasion mechanisms and improve anti-tumor immunity.

The CRISPR-Cas9 system Originally identified as a key defensive mechanism against invading viruses and plasmids in prokaryotic genomes, 19, 20 CRISPR-Cas systems have since been adapted for RNA-programmable genome editing. 21 The most common and best studied of the CRISPR-Cas systems are those that involve the large, multi-domain endonuclease, CRISPR-associated protein 9 (Cas9). The DNA cleavage by Cas9 requires a single-stranded short guide RNA (sgRNA), consisting of a programmable target-specific 20-nt CRISPR RNA (crRNA) base paired to a small non-coding trans-activating crRNA (tracrRNA). Additionally, Cas9 requires a conserved protospacer-adjacent motif (PAM) sequence for activity that maps upstream and adjacent to the crRNA-binding region. The PAM sequence varies depending on the organism of origin and affects the frequency and specificity of the editing process (Table 1) . Upon sequence-specific binding between the crRNA and target DNA, the Cas9 protein is recruited to the PAM sequence via its PAM-interacting domain. After binding, the separation of the target DNA is initiated at the PAM-adjacent nucleation site and double-stranded breaks (DSBs) are produced. The DSBs can be repaired by either homology-directed repair (HDR) or non-homologous end-joining (NHEJ) ( Figure 1A) . 21, 26 The efficiency of genome editing in this way varies greatly and is dependent on several factors, including the nature of the target and delivery method used. Notably, Mussolino et al. 35 reviewed the editing frequencies achieved in the case of the hematopoietic system and found that ex vivo editing is often 10%-30% efficient, whereas in vivo editing varies between 1% and 16%. However, with an optimized delivery system specific for the target cells, the Cas9 editing efficiency can increase up to 80%. 36, 37 The CRISPR-dCas9 system Beyond the success of the CRISPR-Cas9 system, the creation of the nuclease-deactivated Cas9 (dCas9) variant 38 has widened the scope of CRISPR technologies into the field of epigenome engineering. The CRISPR-dCas9 system differs from the wild-type by two mutations (D10A and H840A), which inactivate Cas9's cleavage capacity, while maintaining its RNA-guided DNA-binding specificity. 38 As initially shown with engineered zinc finger proteins, [39] [40] [41] [42] [43] [44] [45] [46] dCas9 can be fused with various effector domains to mediate precise and programmable transcriptional activation or repression, editing of epigenetic marks, and fluorescent tagging of endogenous genes, all without directly editing the genome ( Figure 1B ). [47] [48] [49] [50] [51] [52] Locus-specific transcriptional manipulation in a guide-dependent manner was first achieved by fusing the VP64 (four copies of VP16, a herpes simplex virus transcription factor) recruiter of transcriptional activators, 53-55 as well as Krüppel-associated box (KRAB) recruiter of transcriptional repressors, 38, 53 to dCas9. Since then, more complex arrays of mechanistically distinct effector domains have been described, which greatly improve the capability of dCas9 to induce transcriptional changes. This includes the VPR (tandem fusion of VP64, p65, and Rta domains 56 to generate the hybrid tripartite activator) in the case of gene activation, and the tandem fusion of KRAB with the TRD domain of MeCP2 to produce the dCas9-KRAB-MeCP2 repressor. 57 As the arrays of effectors directly fused to dCas9 increase the size of the resulting protein, which in turn impact the expression of dCas9 and the intracellular delivery, alternative assembly methods have been developed. Notably, aptameric motifs engineered in combination with the gRNA scaffold (such as two copies of an RNA hairpin from the MS2 bacteriophage) were first described in the context of gene activation with the synergistic activation mediator (SAM) system. 58 This approach has been exploited to combine multiple activator domains derived from epigenetic enzymes, such as the catalytic domain of DNA demethylases (e.g., TET1 59,60 ) or histone acetyltransferases (e.g., p300 61 ), to generate high levels of gene activation. Alternatively, repetitive peptide arrays that amplify and recruit specific designer antibody-fusion proteins can be fused to dCas9, as shown in the supernova tagging (SunTag) 62 system. The dCas9-SunTag is based on single-chain variable fragment antibodies and the corresponding epitope, which offers major advantages, including high affinity and recognition of short peptide sequences. This system has previously been adapted to recruit DNA methyltransferases, including DNMT3A 63 and DNMT3L, 64 in order to induce locus-specific repression via DNA methylation, with minimal off-target binding. 65 Importantly, these next-generation dCas9 systems (SAM and SunTag) provide highly specific, effective, and tunable tools for targeted epigenetic manipulation. Lastly, the further development of these technologies to enable simultaneous expression of multiple gRNAs (multiplexed transcriptional manipulation of distinct genes) 66, 67 continues to widen the scope and translatability of dCas9-based epigenetic editing.

In the arena of oncology, these dCas-based tools have demonstrated significant activation of tumor suppressor genes, such as PTEN (in breast cancer and melanoma), 48 MASPIN (in breast and lung cancers), 47, 49 REPRIMO (in breast and gastric cancers), 47 SARI (in colon cancer), 68 and DKK3 (in prostate cancer). 69 Similarly, 70 and liver cancer (GRN). 73 In addition, several works outline that epigenome editing can be highly efficient, having achieved nearly complete gene repression 38 or robust (several fold) gene activation, 56 with minimal off-target effects, which mainly depend on the nature of effector domains used. For the most studied domains, such as VP64 and KRAB, off targets have been shown to be either zero or have negligible effects on non-cognate gene transcription. 74 Finally, whereas Cas9 genome engineering unavoidably results in permanent changes, epigenetic approaches are reversible, circumventing the risk of inducing sequence changes in the target DNA, 71, 75 a key factor in the targeting of tumors harboring high degrees of genetic instability. Moreover, the durability of the epigenetic and transcriptional changes induced by dCas9 editing can vary depending on the specific combination of effectors and may be dependent on the targeted loci. Thus, current research in the field of epigenome engineering faces the challenge to adapt the technology for the manipulation of different loci in diverse cell types with differing chromatin microenvironments. 76 The CRISPR-Cas13 system

The reduced RNA-cleavage efficiency and the likelihood for offtarget effects on host DNA have meant that RNA-targeting orthologs of Cas9 are unlikely to be useful in targeting RNA transcripts directly, such as non-coding RNA (ncRNA), messenger RNA (mRNA), or viral RNA genomes. 77, 78 Moreover, existing RNA interference (RNAi) technologies also exhibit substantial off-target effects. 79 As such, increasing focus is being directed toward the development of novel CRISPR-based technologies utilizing RNAspecific Cas proteins. To date, the most successful of these technologies are those exploiting Cas13, of which four subtypes have been identified thus far: Cas13a (formerly known as C2c2), Cas13b, Cas13c, and Cas13d. 80 In prokaryotes, Cas13 functions as an RNA-guided RNA endonuclease and operates as a defensive mechanism specific for viral RNAs. 81 Unlike Cas9, the Cas13 effector family contains two higher eukaryotes and prokaryotes nucleotidebinding (HEPN) domains as its catalytic effectors, which confer RNase activity ( Figure 1C ). In addition, the Cas13 protein can form a complex with multiple crRNAs to cleave at multiple locations along an RNA transcript with high specificity and offers an alternative gene knockdown method to RNAi technologies ( Figure 1D ). 82, 83 Beyond Cas13's cleavage capability, Zhang and colleagues [84] [85] [86] have pioneered the potential of catalytically inactive Cas13 (dCas13) in several applications. In particular, dCas13 has been fused with fluorescent tags to precisely label target RNA molecules and assess RNAspecific intracellular localization. Similarly, the fusion of dCas13 with deaminase domains from adenosine deaminases specific for RNA (ADARs) has been shown to mediate precise RNA editing to alter full-length transcripts containing pathogenic mutations.

Individually and in combination, Cas9, dCas9, and Cas13 have shown great promise as therapeutic options for multiple diseases, such as cancer, 70,87,88 viral infection, 82, 89, 90 non-viral infection, 91,92 and autoimmunity. 93, 94 In oncology, the ability of CRISPR-Cas to efficiently and specifically knock out (Cas9) or repress (dCas9 and Cas13) pro-tumorigenic genes or transcripts opens new strategies for tumor suppression. [95] [96] [97] Moreover, the same system can be used to introduce (Cas9) or activate (dCas9) important immune-related genes to directly improve the host immune response. 95, 96 Consequently, the capacity of CRISPR-based systems to work individually as therapeutics and in combination with current immunotherapy strategies is developing into an emerging area of interest.

As of March 1, 2020, there have been 21 registered trials utilizing CRISPR-Cas systems aiming to genetically alter human T cells-a key adaptive immune cell type that responds specifically to antigens and is critical in host immunity ( 100 (ClinicalTrials.gov: NCT03399448). Outcomes from these trials are discussed below.

Many tumors, including esophageal squamous cell carcinoma (ESCC) and non-small cell lung cancer (NSCLC), express immune checkpoint PD-1 ligands (PD-L1s) that bind to PD-1 receptors on host T cells to inhibit their proliferation and cytokine production, thereby enabling immunoevasion ( Figure 2 ). [101] [102] [103] To date, anti-PD-1 and anti-PD-L1 monoclonal antibodies have shown success in many cancers, including melanoma, 104 Hodgkin's lymphoma, 105 liver cancer, 106 and lung cancer. 107 However, given the potential of PD-1/PD-L1 inhibitor therapy to result in severe toxicity, 108,109 genetic disruption of PD-1 expression on host T cells is seen as an alternative and potentially safer immunotherapeutic avenue. Jing et al. 98 and Lu et al. 99 independently investigated the safety of PD-1 knockout T cell reinfusion in 17 patients with advanced ESCC and 12 patients with metastatic NSCLC, respectively. In both trials, adoptive cell transfer (ACT) was used, whereby peripheral blood mononuclear cells (PBMCs) were collected, followed by ex vivo CRISPR-Cas9-mediated PD-1 (PDCD1 gene) knockout. The edited PBMCs were then selected, expanded, and reinfused back into each patient. In both studies, the regimen was well tolerated, with no serious (grade 3/4) treatment-related adverse events observed. No complete or partial responses were witnessed in either trial; however, approximately 35% (6/17) of ESCC patients and 18% (2/11; early withdrawal by one patient due to bacterial infection) of NSCLC patients exhibited stable disease. Results by Lu et al. 99 demonstrated that co-transfection of Cas9 and sgRNA plasmid DNA (pDNA) via electroporation resulted in a low median editing efficiency of 5.81% (range, 0.42%-24.85%) in the 12 enrolled patients. In the edited PBMC pool, a median of 99.1% of cells were CD3 positive (range, 95.9%-99.6%), with CD3 + CD8 + T cells accounting for 73.5% (range, 38.5%-93.0%). Whole-genome sequencing at 100-fold coverage targeted toward 2,086 sites determined by Cas-OFFinder 110 detected no true off-target events, which constitute arguably the greatest cause for concern in CRISPR-Cas9 clinical applications. Ultimately, both trials appear to confirm that only minor (grade 1/2) adverse effects, including fatigue, fever, joint pain, and skin rash, were attributed to the treatment, suggesting that CRISPR-Cas9-mediated ACT may be safe for clinical use. Moreover, although therapeutic efficacy was not the focus of these trials, they nonetheless highlight that significant improvements, such as a substantially increased editing efficiency, greater expansion of tumor-reactive T cells, enhanced antigen specificity, and a clearer understanding of the specific T cell subtypes undergoing the editing process, are required in order for an improved patient response. Importantly, both trials support that CRISPR-Cas9-based immunotherapy warrants further clinical investigation.

Results from the first phase I clinical trial to test the safety of multiplex CRISPR-Cas9 in treating patients with refractory cancer (Clinical-Trials.gov: NCT03399448) were published in February 2020 by Stadtmauer et al. 100 Three patients were recruited and CRISPR-Cas9 was applied ex vivo to simultaneously disrupt expression of the PDCD1 gene (PD-1) and the endogenous T cell receptor a/b chain genes (TRAC, TRBC), which encode the T cell surface receptor (TCR) responsible for recognition of antigenic peptides presented in the context of MHC class I and II molecules. Patient CD3 + T cells were further transduced with TCRs specific for tumor antigen (NY-ESO-1) recognition to enhance anti-tumor responses. The same ACT process as above was used, with the engineered T cells being reinfused back into each patient following editing and expansion. Stable disease was seen in two patients, whereas the third patient exhibited disease progression. Importantly, no serious (grade 3/4) adverse events were caused by the treatment, with the re-infusion process and persistence of transduced cells being well tolerated by all patients. Unlike the plasmid-based delivery method used by Lu et al., 99 Stadtmauer et al. 100 utilized electroporation of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 loaded with equimolar mixtures of the three sgRNAs targeting each gene. In this case, the editing efficiency was approximately 20% for PDCD1, 45% for TRAC, and 15% for TRBC. Although RNP delivery shows significant editing improvements compared to plasmid-based systems, 99 incomplete editing of TRAC/TRBC genes has previously been reported to result in mispairing and/or competing for expression between the transgenic TCR and endogenous TCR. 111 Additionally, iGUIDE, 112 a variant of GUIDE-seq (genome-wide unbiased identification of DSBs enabled by sequencing), 113 determined three significant off-targets. Unknown studies denote those whose last known status was recruiting, not yet recruiting, or active, but had passed the completion date, and the status had not been last verified within the past 2 years.

The first, caused by the TRAC sgRNA, affected the transcriptional unit of chloride intracellular channel 2 (CLIC2) and was deemed acceptable, as it is not reported to be expressed in T cells. The remaining two off-targets, within the genes encoding zinc finger protein 609 (ZFN609) and long intergenic non-protein coding RNA 377 (LINC00377), were attributed to the TRBC sgRNA. Both off-targets were also deemed by the authors as acceptable due to their minimal impact on the gene (ZFN609) and unknown role to date (LINC00377). Despite the small sample size, Stadtmauer et al. 100 supported that multiplexed CRISPR-Cas9-directed ACT is feasible and appears to be safe for patients. Additionally, their trial reinforces that off-targeting continues to be an important consideration in any clinical application of CRISPR, and high-throughput sequencing technologies should be used to enable their identification. 114 Lastly, although the high editing efficiency achieved in this trial using RNP delivery verifies the established pre-clinical success of RNPs as the preferred CRISPR delivery system, 115, 116 further improvements to ex vivo CRISPR editing are clearly needed to see an improved patient response. Namely, complete editing of TRAC/TRBC genes to eliminate the possibility of TCR mispairing, a detailed understanding of the specific T cell subsets undergoing editing, and extensive functional elucidation of off-targets and their effects (particularly, novel targets such as LINC00377) should be explored in future studies.

Using CRISPR-Cas9-based gene editing to target immune checkpoint pathways, such as the PD-1/PD-L1 axis, in combination with re-engineering the TCR for tumor specificity is expected to be an effective strategy to improve immune recognition and, ultimately, to promote the elimination of cancer cells. Moreover, use of these technologies is likely to complement and enhance patient responsiveness and/or serve as an alternative to monoclonal antibody treatment that targets immune checkpoint inhibitors (ICIs), such as PD-1 or CTLA-4, which have been effective in only a subset of cancers. [117] [118] [119] Aside from the suitable safety documented in the above CRISPR-Cas9-based anti-tumor immunotherapy clinical trials, the small sample sizes and high variability in survival of individual patients limit definitive conclusions on the efficacy of these treatments. However, with a number of current CRISPR-based trials targeted toward boosting the immune response via manipulation of immune-related genes ( Table 2) , more conclusive results are likely to be seen during the coming months.

While major modalities of cancer immunotherapy, such as ICIs and ACT, have revolutionized cancer treatment, not all patients respond to these therapies. 107, 120 Often, durable immune responses occur in patients with immunogenic ("hot") tumors, characterized by infiltrating CD4 + and CD8 + T cells and accumulation of pro-inflammatory mediators. 121 However, non-immunogenic ("cold") tumors that lack these components have significantly reduced response rates, although some patients still respond, and cold tumors may require conversion into a more hot-like phenotype in order to achieve a better outcome. 122 The CRISPR-dCas9-mediated epigenetic modulation of tumor immunogenicity represents one such conversion method.

In this approach, dCas9-mediated transcriptional activation of pro-immunogenic genes, or repression of genes involved in tumor immunoevasion and immunosuppression, may enhance tumor immunogenicity. 122 Studies using these methods have shown success in pre-clinical testing and hold great potential for novel immunotherapeutics, as discussed below. Using either of these approaches has been shown to improve the killing capacity of immune effector cells. CRISPR-dCas9 linked to Act can also be used to directly upregulate the genes responsible for antigen presentation, MHC class I (iii) and MHC class II (iv). Increased presentation of antigens improves the likelihood of recognition and elimination via CD8 + T cells and CD4 + T cells, particularly those re-engineered ex vivo to recognize specific tumor antigens.

In a seminal 2019 publication by Wang et al., 123 a multiplexed dCas9-SAM system was harnessed to induce a genome-scale simultaneous upregulation of endogenous pro-immunogenic genes in triple-negative breast cancer (TNBC) E0771 cells, in order to improve tumor immunogenicity. Given their role as inducers of T cell proliferation and activation, a particular focus was directed toward the upregulation of CD70, CD80, CD86, IFNa4, IFNb1, and IFNg genes. CD70, present on antigen-presenting cells (APCs; dendritic cells, B cells, and macrophages) and some T cells, is the cognate ligand for the tumor necrosis factor (TNF) receptor family member CD27 on T cells, and it provides an essential co-stimulatory signal for T cell activation. 124, 125 Similarly present on APCs and some T cells, CD80 and CD86 are cognate ligands for the co-stimulatory receptor CD28 on T cells and also play a critical role in T cell activation. 126, 127 Type I (a and b) and type II (g) interferons (IFNs) are potent cytokines that bind to the ubiquitously expressed IFNa receptor (IFNaR) and IFNgR, respectively. Most cell types can produce IFNb, whereas IFNa is predominantly produced by plasmacytoid dendritic cells and IFNg is chiefly secreted by activated T cells and natural killer (NK) cells. Together, type I and II IFN signaling promotes innate and adaptive immunity in a variety of ways, including activation of antigen presentation and chemokine production, enhanced antibody generation, and stimulation of T cell and NK cell cytotoxicity. 128, 129 The transcriptional upregulation mediated by dCas9-SAM resulted in amplified presentation of a model antigen (ovalbumin) in vitro. Moreover, the edited E0771 cells transplanted into syngeneic immunocompetent (C57BL/6J) mice demonstrated significantly reduced tumor volume in vivo compared to both immunodeficient (nude and Rag-deficient) mice and CD4 + and CD8 + T cell-depleted immunocompetent mice. Additionally, Wang et al. 123 combined their activation system with anti-CTLA4 monoclonal antibodies, which substantially increased the efficacy of anti-CTLA4 therapy, with complete tumor regression of established tumors. As expected, the increased tumor immunogenicity via dCas9-mediated transcriptional activation of pro-immunogenic genes increased CD4 + and CD8 + T cell infiltration into the tumor, thereby improving anti-tumor immunity.

The findings were also supported by Liu et al.'s 130 earlier work from 2017, where HeLa cells edited using the same dCas9-SAM system to overexpress the IFNg gene exhibited enhanced apoptosis, inhibited proliferation, and overall reduced tumor volume when implanted in immunodeficient (severe combined immunodeficiency [SCID]) mice. Collectively, both works provide a proof of concept that manipulating the transcriptome of tumors in favor of a proimmunogenic phenotype can greatly improve the anti-tumor immune response and the response to ICIs. Moreover, the activation of endogenous cytokines, such as IFNg, within the tumor is likely to be an excellent starting point for this novel approach of enhancing tumor immunogenicity.

The TGF-b signaling axis has been of interest owing to its dual role in both tumor suppression and progression. 131, 132 When functioning as an oncogenic activator, TGF-b signaling induces an immunosuppressive response, 133 which is further potentiated by reciprocal positive regulatory interactions with Notch and Hippo signaling. 134 Recently, microRNA-524 (miR-524) has been shown to silence the TGF-b, Notch, and Hippo pro-tumorigenic signaling pathways simultaneously by suppression of SMAD2, HES1, and TEAD1 genes, respectively, making it an attractive target for cancer immunotherapy. 135 As such, in 2019, Liu et al. 136 aimed to explore the potential of dCas9-VP64-mediated miRNA-524 transcriptional activation as a cancer immunotherapy strategy to inhibit TGF-b/Notch/Hippo signaling in vivo. To this end, a pH-responsive multistage delivery nanoparticle (MDNP) was developed to deliver pDNA encoding the CRISPR-dCas9 system and sgRNA biomolecular components targeting the miR-524 locus. The systemic injection of MDNP/ dCas9-miR-524 into tumor-bearing immunodeficient (nude) mice with TNBC MDA-MD-231 xenograft evidenced reduced SMAD2, HES1, and TEAD1 gene and protein expression, significantly inhibiting tumor growth and higher levels of tumor apoptosis. Although the direct impact of miR-524 activation on tumor immunogenicity was not explored in this study, inhibition of the TGF-b axis is well recognized to improve immune cell infiltration as a consequence of the reduction in immunosuppression, tumor migration, and angiogenesis. 137, 138 Altogether, Liu et al.'s 136 work provides a strong rationale for further exploration of miR-524 in cancer immunotherapy and highlights the potential of CRISPR-dCas9 technologies to inhibit immunosuppressive pathways for an improved antitumor response.

Although not applied in pre-clinical studies, a number of early works also suggest that dCas9-based methods can help circumvent other major tumor immunoevasion mechanisms. For instance, impairment of Treg immunosuppressive function by dCas9-KRAB-mediated forkhead box P3 (FoxP3) transcriptional repression, 139,140 dCas9-SAM-mediated enhancement of neoplastic transformation markers, such as MICA, 141, 142 and dCas9-KRAB-directed repression of immunosuppressive cytokine receptors 143 have demonstrated that dCas9mediated epigenetic engineering has potential in multiple facets of cancer immunotherapy. Ultimately, dCas9-mediated epigenetic editing to improve tumor immunogenicity and/or boost the host's antitumor immune response opens new therapeutic avenues, particularly in combination with ICI or ACT therapies (Figure 2 ).

Significant attention has been directed toward applying CRISPR-Cas13 (RNA-targeting) technology as a diagnostic in the detection of low-frequency cancer somatic mutations 144 and in RNA editing. 85, 145 Additionally, the CRISPR-Cas13 system has demonstrated highly efficient and specific knockdown of oncogenic mutant drivers, 146 gene fusions, 88 and ncRNA 147 transcripts in vitro with minimal off-targets, highlighting its potential for use as an RNAbased therapeutic tool. More recently, the potential of Cas13-based gene suppression in cancer immunotherapy has drawn attention due to its growing success in pre-clinical studies, as explored below.

As discussed above, CRISPR-mediated PD-1 knockout T cells have shown acceptable toxicity profiles, yet their efficacy remains limited. Moreover, although PD-1 knockout therapies largely focus on T cells, other important immune effectors, such as NK cells, are also inhibited by the PD-1/PD-L1 axis. 148 Therefore, as opposed to targeting PD-1 expression on specific cell types, Zhang et al. 149 explored the capability of CRISPR-Cas13a to silence PD-L1 expression at the tumor cell surface to improve overall anti-tumor immunity. To this end, pDNA encoding Cas13a and crRNAs targeting PD-L1 transcripts were systemically delivered in vivo using a pH and hydrogen peroxide (H 2 O 2 )-responsive dual-locking nanoparticle (DLNP). Following injection of DLNP/Cas13-crRNA into melanoma B16F10-bearing immunocompetent mice, significant tumor growth suppression and improved survival was observed. Furthermore, a significant reduction in TGF-b and elevation in IFNg, TNF-a (predominantly secreted by macrophages and promotes T cell activation), IL-2 (secreted by activated T cells and promotes T cell proliferation), and IL-12 (secreted by activated APCs to activate NK cells and induce T cell differentiation) levels were seen in treated tumors, suggesting activation of antitumor immunity. Knockdown of PD-L1 by Cas13 also increased the number of CD8 + tumor-infiltrating T cells, reduced the number of myeloid-derived suppressor cells (MDSCs), and induced a tumorassociated macrophage polarization from a tumor-promoting M2like (CD206 hi CD11b + F4/80 + ) to a more anti-tumor M1-like (CD80 hi CD11b + F4/80 + ) phenotype in the tumor microenvironment. This work suggests that Cas13a can successfully suppress tumor PD-L1 expression and aid in eliciting an effective anti-tumor immune response ( Figure 2 ).

The VEGF/VEGF receptor pathway constitutes one of the most promising therapeutic targets due to its significant immunosuppressive role in the tumor and surrounding microenvironment. Previously, obstruction of VEGF receptor 2 (VEGFR2) 150 and its downstream signaling pathways, BCL-2 and Survivin, 151 via small-molecule inhibitors or neutralizing antibodies has shown success in blocking tumor growth and prolonging survival. However, limitations, such as temporary efficacy, treatment resistance, and adverse complications, have justifiably limited their clinical use. With this in mind, Fan et al. 152 explored the capacity of the Cas13a system to target VEGFR2, BCL-2, and Survivin transcripts simultaneously in tumor cells. Plasmid DNA containing Cas13a and crRNA tandem sequences designed to target VEGFR2/BCL-2/Survivin transcripts were encapsulated in a dual-component liposome system coated in VEGFR2 monoclonal antibodies. The Cas13a/liposome was then perfused via intravesical administration into bladder cancer 5637 cell-bearing immunodeficient (nude) mice, resulting in significantly reduced transcription levels of VEGFR2/BCL-2/Survivin and inhibited tumor growth. It is well established that inhibition of the VEGF/VEGFR axis reduces angiogenesis and Treg/MDSC accumulation, thereby promoting immune cell infiltration into the tumor. [153] [154] [155] These findings therefore support that Cas13a represents an emerging precision medicine platform for the inhibition of tumor growth by targeting pro-oncogenic signaling pathways, such as VEGF/VEGFR, in a highly selective manner.

More recent studies have shown that dCas13-fusion (RNA editing) proteins can also achieve efficient knockdown of endogenous RNA transcripts by catalyzing the demethylation of m 6 A (N6-methyladenosine), 156 or by the degradation of m 6 A-marked RNA, 157 resulting in gene suppression. These early works suggest that dCas13-based technologies targeted toward m 6 A modifications may offer an alternative to Cas13-mediated alteration of post-transcriptional RNA fate. Although more investigation is required to explore the efficacy and safety of Cas13 in cancer immunotherapy, studies support that Cas13 has the potential to function as a therapeutic through its targeted manipulation of tumor immunogenicity (Figure 2 ).

To date, many reviews have discussed the key considerations surrounding use of CRISPR in treating human diseases. For further details on the concerns surrounding use of CRISPR-based therapeutics in a general sense, we refer to excellent previously published reviews. 74, [158] [159] [160] [161] In this section, however, we focus specifically on the concerns associated with CRISPR use in cancer immunotherapy.

Given that CRISPR systems are derived from common human pathogens, such as Streptococcus pyogenes and Staphylococcus aureus, the presence of pre-existing immunity from prior exposure remains a major concern in the safety and efficacy of these technologies. Principally, in the context of cancer immunotherapy where potent immune activation is the goal, the extent of anti-Cas antibodies and/or T cells that may interfere with CRISPR-based treatment needs to be elucidated. 119, 162 Recently, three studies have shown that both antibodies and T cells against Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) are indeed present in many individuals, supporting that further investigation is required. First, Simhadri et al. 163 highlighted that approximately 2.5% and 10% of donors (a predominantly white cohort from the United States) tested positive for anti-SpCas9 and anti-SaCas9 antibodies in human sera, respectively, by enzyme-linked immunosorbent assay (ELISA). Second, Wagner et al. 164 showed using flow cytometry that approximately 96% of donors (ethnicity unknown) had SpCas9-reactive peripheral CD4 + and CD8 + T cells as measured by CD137 expression. Lastly, Charlesworth et al. 165 identified using PBMCs that approximately 58% and 78% of donors (a predominantly white cohort from the United States) had antibodies against SpCas9 and SaCas9 detected using ELISA, respectively. Moreover, approximately 67% and 78% of donors were positive for SpCas9-and SaCas9-reactive CD4 + /CD8 + T cells, respectively, as confirmed by IFNg enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and/or surface expression of CD137 or CD154. Although these studies complement one another in highlighting the prevalence of pre-existing immunity to Cas9, further research is still required to elucidate whether the presence of anti-Cas antibodies or anti-Cas T cells are at biologically relevant levels and to what extent they may impact CRISPR-based therapy.

Ultimately, if in healthy hosts the potential exists for an anti-CRISPR immune response to impede the efficacy of CRISPR-based therapeutics, this is likely to be further exacerbated in patients undergoing CRISPR/ICI or CRISPR/ACT combination cancer immunotherapy where effector immune cells are in an activated state. In order to circumvent this issue for Cas9 genetic editing, it has been proposed that transient Cas9 expression ex vivo outside of direct immune contact and reinfusion into the host once the Cas9 protein has been cleared may be a solution. 165 This may help to explain why CRISPR-based clinical trials to date-all using ex vivo editinghave not shown adverse Cas9-reactive T cell responses. However, the likelihood of producing anti-Cas9 memory T cells during the ex vivo editing process is still unknown, and moreover this ex vivo method cannot be applied to dCas9 or Cas13 systems where in vivo use is vital. In these cases, preclinical assessment of Cas immunogenicity or specific delivery methods that do not allow for CRISPR-immune interactions until cargo release may be required. Examples of successful delivery systems designed with this is mind include the MDNP 136 and DLNP 149 methods discussed in the pre-clinical dCas9 and Cas13 sections, respectively. These delivery systems are also necessary to ensure that the CRISPR cargo is delivered directly to the target tissue. For instance, the release of CRISPR-dCas9 with sgRNAs targeting pro-immunogenic genes in unintended tissues may elicit an adverse pro-inflammatory and/or autoimmune response. Ultimately, averting an anti-CRISPR immune response will likely require a joint approach of checking for pre-existing immunity and selecting for tissue-specific, multi-layered, non-immunogenic delivery systems.

One of the key benefits of CRISPR-based systems is their capability to multiplex crRNAs and, thus, to regulate multiple targets simultaneously. For cancer immunotherapy, it is highly likely that clinical benefit will only be achieved through multiplexing, as multiple genetic elements require editing. However, as the number of target sites increases, there is also a subsequent increase in the competition between crRNAs to guide the Cas protein to their target site. Competition between crRNAs, termed retroactivity, 166 results in a decrease in the performance of CRISPR-Cas and severely hinders the ability to predict crRNA targeting efficiency. This presents a major obstacle in cancer immunotherapy, as it is essential that maximum gene editing and transcriptional regulation is achieved. In other contexts, retroactivity has been mitigated by using conditional crRNAs, whose activity is dependent on the presence or absence of an RNA trigger. 167 However, this is unlikely to serve in cancer immunotherapy, as simultaneous targeting is required. An alternative method more applicable to overcoming retroactivity in cancer immunotherapy lies in the exploitation of target hierarchy. In this approach, targets of high priority are allocated multiple crRNAs, while lower priority targets are allocated only a single crRNA. 66 This method may ensure that all sites are targeted, but those of high importance are emphasized. Furthermore, it is still unclear as to how many crRNAs can be expressed in vivo for an effective multiplexing strategy, while maintaining editing efficacy at all target sites. This knowledge is fundamental to achieving the desired effectiveness of CRISPR-based therapies. In the case of cancer immunotherapy, it is expected that not all target sites will have the same impact on tumor immunogenicity or immune effector cytotoxicity. Also, inherent genetic and/or epigenetic differences between patients at specific target sites is highly likely. Therefore, effective multiplexed strategies that target these different loci is essential.

Altogether, if pre-existing CRISPR immunity and retroactivity can be mitigated via improved delivery systems and crRNA prioritization/ customization to particular patients, then CRISPR-based cancer immunotherapy may yet develop to become an effective clinical treatment.

The ability of CRISPR-based technologies to efficiently modify specific loci at the genetic and epigenetic level provides novel opportunities to reprogram the immune response for improved tumor elimination. To date, CRISPR-Cas9 genetic editing has successfully improved the killing capacity of T cells via the elimination and/or modification of key cell surface molecules, such as PD-1 and the TCR. However, although shown to be safe when used ex vivo, Cas9's potential to bind to non-target DNA sequences (off-targets), thereby causing permanent genomic instability or disruption of otherwise normal host genes, has consistently remained a major concern. 168,169 Alternatively, the development of dCas9-and Cas13-based systems that work transiently, are reversible, and eliminate the risk of long-lasting consequences are expected to be safer, and therefore more clinically relevant, for in vivo use. Technologies such as dCas9 in particular show significant advantages, as it mediates straightforward upregulation or downregulation of transcription and editing of epigenetic marks within the same platform, which significantly expand its applications in cancer therapeutics. Moreover, these systems are increasingly being developed as inducible structures allowing for precise control of epigenetic editing at will. 170 Still, safe and precise delivery mechanisms are yet to be addressed in a comprehensive way, and the potential for adverse immune reactions challenge clinical CRISPR applications. Altogether, based on safety and versatility, epigenetic editing platforms such as dCas9 and Cas13 are likely to overtake Cas9 genetic editing platforms in clinical applications.

Future applications of CRISPR-based technologies in the immunotherapy field are likely to expand heavily on epigenetic editing by dCas9 or Cas13. Epigenetically reprogramming immune evasion mechanisms, such as rescuing MHC class I/II expression, halting MICA/B shedding, and repressing the activation of suppressive cytokines, is an attractive therapeutic avenue, as doing so has the potential to greatly improve host immunity. Furthermore, the expanding knowledge of the role of distinct immune checkpoint inhibitors will offer grounds for the identification of additional checkpoint molecules to be edited in multiplexed CRISPR applications. Selective epigenetic targeting of immune checkpoints in effector immune cells or at the tumor cell surface to improve in vivo immune activation and efficacy is likely to improve patient outcomes in the clinic. Other potential applications may involve the epigenetic manipulation of cells used in ACT aiming at enhancing intra-tumoral recruitment and persistence of these cells through the upregulation of relevant chemokine receptors, such as CXCR2 and CXCR3, which greatly improve T cell localization and migration to tumors. [171] [172] [173] In principle, this strategy could be combined with epigenetic upregulation of the genes encoding for the corresponding chemokine ligands on tumor cells, thus facilitating the treatment of cold-like tumors, which are poorly amenable by current immunotherapies. Similarly, recent studies have highlighted the complexity in the development of exhausted T cells and NK cells (failure to produce cytokines, lack of proliferation, and high expression of inhibitory receptors), 174-177 with significant epigenetic changes identified in chronically stimulated NK cells 178 and T cells that failed to respond to anti-PD-1 therapy. 179, 180 Reinvigoration of exhausted and dysfunctional cell subsets using targeted epigenetic CRISPR editing of key genes, such as TCF1 (transcription factor encoded by TCF7 crucial for T cell persistence and NK cell survival), [181] [182] [183] may assist in improving patient responses to ICI therapies and overall anti-tumor immunity. Importantly, the extent to which CRISPR-based technologies can alter the tumor landscape is still unknown; however, through multiplexed approaches, it is hopeful that durable immune responses can be achieved. Alhough in its infancy, precise epigenetic editing by dCas9 and Cas13 is likely to become a powerful tool in both basic scientific research and clinical application, especially when combined with Cas9-mediated genetic editing.

Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival

Two FOXP3 + CD4 + T cell subpopulations distinctly control the prognosis of colorectal cancers

Multiple genetic alterations cause frequent and heterogeneous human histocompatibility leukocyte antigen class I loss in cervical cancer

Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer

An evolutionarily conserved function of polycomb silences the MHC class I antigen presentation pathway and enables immune evasion in cancer

Natural killer cell cytotoxicity is suppressed by exposure to the human NKG2D ligand MICA*008 that is shed by tumor cells in exosomes

Shedding of endogenous MHC class I-related chain molecules A and B from different human tumor entities: heterogeneous involvement of the "a disintegrin and metalloproteases" 10 and 17

Inhibition of MICA and MICB shedding elicits NK-cell-mediated immunity against tumors resistant to cytotoxic T cells

T cell surveillance of oncogene-induced prostate cancer is impeded by T cell-derived TGF-b1 cytokine

TGF-b and VEGF cooperatively control the immunotolerant tumor environment and the efficacy of cancer immunotherapies. JCI Insight 1, e85974. supports an adaptive resistance mechanism of immune escape

PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity

Enhancement of antitumor immunity by CTLA-4 blockade

Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies

The next generation of CRISPR-Cas technologies and applications

CRISPR provides acquired resistance against viruses in prokaryotes

Small CRISPR RNAs guide antiviral defense in prokaryotes

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity

Structure and engineering of Francisella novicida Cas9

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

Short motif sequences determine the targets of the prokaryotic CRISPR defence system

RNA-guided editing of bacterial genomes using CRISPR-Cas systems

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

Phage response to CRISPR-encoded resistance in Streptococcus thermophilus

Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis

In vivo genome editing using Staphylococcus aureus Cas9

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition

Crystal structure of the minimal Cas9 from Campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 systems

In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Genome and epigenome editing to treat disorders of the hematopoietic system

One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering

CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy

Repurposing CRISPR as an RNA-guided platform for sequencespecific control of gene expression

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

Modulation of drug resistance by artificial transcription factors

Reprogramming epigenetic silencing: artificial transcription factors synergize with chromatin remodeling drugs to reactivate the tumor suppressor mammary serine protease inhibitor

Suppression of breast tumor growth and metastasis by an engineered transcription factor

Reactivation of MASPIN in non-small cell lung carcinoma (NSCLC) cells by artificial transcription factors (ATFs)

Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer

Epigenetic reprogramming of cancer cells via targeted DNA methylation

Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/ dCas9 system

Activating PTEN tumor suppressor expression with the CRISPR/dCas9 system

Tumour suppression by targeted intravenous non-viral CRISPRa using dendritic polymers

Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing

Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system

CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes

RNAguided gene activation by CRISPR-Cas9-based transcription factors

CRISPR RNA-guided activation of endogenous human genes

Highly efficient Cas9-mediated transcriptional programming

An enhanced CRISPR repressor for targeted mammalian gene regulation

Genomescale transcriptional activation by an engineered CRISPR-Cas9 complex

A CRISPR-based approach for targeted DNA demethylation

Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

A protein-tagging system for signal amplification in gene expression and fluorescence imaging

DNA epigenome editing using CRISPR-Cas SunTagdirected DNMT3A

Engineering of effector domains for targeted DNA methylation with reduced offtarget effects

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts

Multiplexed and tunable transcriptional activation by promoter insertion using nuclease-assisted vector integration

Targeted demethylation of the SARI promotor impairs colon tumour growth

CRISPR-mediated reactivation of DKK3 expression attenuates TGF-b signaling in prostate cancer

Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression

Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner

Transcriptional dysregulation of MYC reveals common enhancer-docking mechanism

Epigenetic targeting of granulin in hepatoma cells by synthetic CRISPR dCas9 epi-suppressors

Epigenome engineering: New technologies for precision medicine

Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner

Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase

Cas9-mediated targeting of viral RNA in eukaryotic cells

RNAdependent RNA targeting by CRISPR-Cas9

Expression profiling reveals off-target gene regulation by RNAi

Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and Influenza

Programmable inhibition and detection of RNA viruses using Cas13

RNA targeting with CRISPR-Cas13

RNA editing with CRISPR-Cas13

A cytosine deaminase for programmable single-base RNA editing

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

Effective RNA knockdown using CRISPR-Cas13a and molecular targeting of the EML4-ALK transcript in H3122 lung cancer cells

Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus

Specific reactivation of latent HIV-1 by dCas9-SunTag-VP64-mediated guide RNA targeting the HIV-1 promoter

CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants of Plasmodium falciparum invasion

Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum. Proc. Natl. Acad. Sci. USA

CRISPR/cas9 mediated knockout of an intergenic variant rs6927172 identified IL-20RA as a new risk gene for multiple autoimmune diseases

CRISPR-on system for the activation of the endogenous human INS gene

Applications of the CRISPR-Cas9 system in cancer biology

CRISPR/Cas mediated epigenome editing for cancer therapy

CRISPR-Cas13 precision transcriptome engineering in cancer

Safety and activity of programmed cell death-1 gene knockout engineered t cells in patients with previously treated advanced esophageal squamous cell carcinoma: An open-label, single-arm phase I study

Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer

CRISPR-engineered T cells in patients with refractory cancer

Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death

PD-1 blockade inhibits hematogenous spread of poorly immunogenic tumor cells by enhanced recruitment of effector T cells

Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer

Improved survival with ipilimumab in patients with metastatic melanoma

Treatment of relapsed or refractory classical Hodgkin lymphoma with the anti-PD-1, tislelizumab: results of a phase 2, single-arm, multicenter study

Camrelizumab in patients with previously treated advanced hepatocellular carcinoma: A multicentre, open-label, parallel-group, randomised, phase 2 trial

Five-year overall survival for patients with advanced non-small-cell lung cancer treated with pembrolizumab: Results from the phase I KEYNOTE-001 study

Incidence of programmed cell death 1 inhibitor-related pneumonitis in patients with advanced cancer: A systematic review and meta-analysis

Pneumonitis in patients treated with anti-programmed death-1/programmed death ligand 1 therapy

Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

Orthotopic replacement of T-cell receptor a-and b-chains with preservation of near-physiological T-cell function

iGUIDE: An improved pipeline for analyzing CRISPR cleavage specificity

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

Offtarget effects in CRISPR/Cas9-mediated genome engineering

Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing

Highly efficient RNAguided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Pooled analysis of longterm survival data from phase II and phase III trials of ipilimumab in unresectable or metastatic melanoma

Association of pembrolizumab with tumor response and survival among patients with advanced melanoma

Immunogenicity of CAR T cells in cancer therapy

Long-lasting complete responses in patients with metastatic melanoma after adoptive cell therapy with tumor-infiltrating lymphocytes and an attenuated IL2 regimen

PD-1 blockade induces responses by inhibiting adaptive immune resistance

Turning cold into hot: Firing up the tumor microenvironment

Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity

Constitutive CD27/CD70 interaction induces expansion of effector-type T cells and results in IFNg-mediated B cell depletion

CD70 expression determines the therapeutic efficacy of expanded human regulatory T cells

CD80 and CD86 differentially regulate mechanical interactions of T-cells with antigen-presenting dendritic cells and B-cells

CD80 + and CD86 + B cells as biomarkers and possible therapeutic targets in HTLV-1 associated myelopathy/tropical spastic paraparesis and multiple sclerosis

Low-dose IFN-g induces tumor MHC expression in metastatic malignant melanoma

IFN-a/b enhances BCR-dependent B cell responses

Specific expression of interferon-g induced by synergistic activation mediator-derived systems activates innate immunity and inhibits tumorigenesis

Transforming growth factor b signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis

High TGFb-Smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene

TGFb attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells

Notch and TGFb form a positive regulatory loop and regulate EMT in epithelial ovarian cancer cells

EGFR/c-myc axis regulates TGFb/Hippo/Notch pathway via epigenetic silencing miR-524 in gliomas

Multistage delivery nanoparticle facilitates efficient CRISPR/ dCas9 activation and tumor growth suppression in vivo

Differential inhibition of the TGF-b signaling pathway in HCC cells using the small molecule inhibitor LY2157299 and the D10 monoclonal antibody against TGF-b receptor type II

Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients

Cutting edge: CRISPR-based transcriptional regulators reveal transcription-dependent establishment of epigenetic memory of Foxp3 in regulatory T cells

CRISPRa-mediated FOXP3 gene upregulation in mammalian cells

Intragenic transcriptional interference regulates the human immune ligand MICA

Transcriptional activation of the MICA gene with an engineered CRISPR-Cas9 system

CRISPR-based epigenome editing of cytokine receptors for the promotion of cell survival and tissue deposition in inflammatory environments

Nucleic acid detection with CRISPR-Cas13a/C2c2

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment

CRISPR-Cas13-mediated knockdown of lncRNA-GACAT3 inhibited cell proliferation and motility, and induced apoptosis by increasing p21, Bax, and E-cadherin expression in bladder cancer

The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: A therapeutic target for CT-011, a novel monoclonal anti-PD-1 antibody

Dual-locking nanoparticles disrupt the PD-1/PD-L1 pathway for efficient cancer immunotherapy

Discovery of potent VEGFR-2 inhibitors based on furopyrimidine and thienopyrimidne scaffolds as cancer targeting agents

Survivin is critically involved in VEGFR2 signaling-mediated esophageal cancer cell survival

A multifunction lipid-based CRISPR-Cas13a genetic circuit delivery system for bladder cancer gene therapy

VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer

VEGF suppresses T-lymphocyte infiltration in the tumor microenvironment through inhibition of NF-kB-induced endothelial activation

Sulforaphane exerts anti-angiogenesis effects against hepatocellular carcinoma through inhibition of STAT3/HIF-1a/VEGF signalling

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Targeted m6A reader proteins to study epitranscriptomic regulation of single RNAs

CRISPR/Cas9-based engineering of the epigenome

Advancements and obstacles of CRISPR-Cas9 technology in translational research

Ready for repair? Gene editing enters the clinic for the treatment of human disease

Innovative precision gene-editing tools in personalized cancer medicine

AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9

Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population

High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population

Identification of preexisting adaptive immunity to Cas9 proteins in humans

Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design

Conditional guide RNAs: Programmable conditional regulation of CRISPR/Cas function in bacterial and mammalian cells via dynamic RNA nanotechnology

High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

Complex transcriptional modulation with orthogonal and inducible dCas9 regulators

Redirecting migration of T cells to chemokine secreted from tumors by genetic modification with CXCR2. Hum

Transduction of tumorspecific T cells with CXCR2 chemokine receptor improves migration to tumor and antitumor immune responses

Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints

Network analysis reveals centrally connected genes and pathways involved in CD8 + T cell exhaustion versus memory

CD8 + T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1

Rapid development of exhaustion and down-regulation of eomesodermin limit the antitumor activity of adoptively transferred murine natural killer cells

High NKG2A expression contributes to NK cell exhaustion and predicts a poor prognosis of patients with liver cancer

Chronic stimulation drives human NK cell dysfunction and epigenetic reprograming

Subsets of exhausted CD8 + T cells differentially mediate tumor control and respond to checkpoint blockade

Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade

Defining T cell states associated with response to checkpoint immunotherapy in melanoma

Intratumoral Tcf1 + PD-1 + CD8 + T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy

The transcription factor Tcf1 contributes to normal NK cell development and function by limiting the expression of granzymes

The authors declare no competing interests.