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Review
. 2020 Sep 29:11:2062.
doi: 10.3389/fimmu.2020.02062. eCollection 2020.

CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer

Mohammadreza Azangou-Khyavy  1 , Mobina Ghasemi  1 , Javad Khanali  1 , Melika Boroomand-Saboor  1 , Monire Jamalkhah  2 , Masoud Soleimani  3 , Jafar Kiani  4   5
Affiliations
Review

CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer

Mohammadreza Azangou-Khyavy et al. Front Immunol. .

Abstract

The clustered regularly interspaced short palindromic repeats system has demonstrated considerable advantages over other nuclease-based genome editing tools due to its high accuracy, efficiency, and strong specificity. Given that cancer is caused by an excessive accumulation of mutations that lead to the activation of oncogenes and inactivation of tumor suppressor genes, the CRISPR/Cas9 system is a therapy of choice for tumor genome editing and treatment. In defining its superior use, we have reviewed the novel applications of the CRISPR genome editing tool in discovering, sorting, and prioritizing targets for subsequent interventions, and passing different hurdles of cancer treatment such as epigenetic alterations and drug resistance. Moreover, we have reviewed the breakthroughs precipitated by the CRISPR system in the field of cancer immunotherapy, such as identification of immune system-tumor interplay, production of universal Chimeric Antigen Receptor T cells, inhibition of immune checkpoint inhibitors, and Oncolytic Virotherapy. The existing challenges and limitations, as well as the prospects of CRISPR based systems, are also discussed.

Keywords: CAR T cell therapy; CRISPR; cancer immunotherapy; cancer treatment; gene therapy; genome-wide screening assays; oncolytic virotherapy.

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Figures

FIGURE 1
FIGURE 1
Applications of CRISPR technology in multiple aspects of cancer treatment.
FIGURE 2
FIGURE 2
ZFNs, TALENs, and CRISPR-based genome editing. (A) ZFNs and TALENs are nucleases that operate based on protein-DNA interactions. Assembling Zinc Finger Motifs (ZFMs) and TALEs into larger complexes increases specificity. (B) CRISPR/Cas9 binds to DNA under the guidance of single guide RNA (sgRNA). sgRNA is a chimeric RNA constructed by fusing crRNA and tracrRNA to simplify the guidance system. As a result, Cas9-sgRNA is the most extensively used system in CRISPR based applications (23). For DNA recognition, many CRISPR systems also need Protospacer Adjacent Motif sequence (PAM) adjacent to the crRNA target site. PAM sequences are specific to each type of nuclease (e.g., NGG sequence is specific to SpCas) (21, 198). (C) Double-Stranded Breaks (DSBs) created by Cas9 in DNA structure activate two DNA repair pathways: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair pathways (HDR). NHEJ results in random insertions or deletions at the target site, so it involves knocking out genes in CRISPR-based applications. HDR is a precise pathway that repairs target DNA breakage by using a homologous donor DNA. This pathway takes part in techniques that need more precise genome editing, like insertion or deletion of the desired DNA fragment (199, 200).
FIGURE 3
FIGURE 3
dCas applications: beyond genome editing. (A) dCas9-KRAB has been engineered by fusing the KRAB transcription repressor domain to dCas9 (201). (B) VP64, as a transcription activator, has been fused to dCas9 to activate a specific gene’s transcription (151, 152, 202, 203). (C) Recruiting multiple transcriptional activators shows synergistic effects that have led to engineering many systems, such as VPR by in-tandem fusing of both p65 and Rta to VP64 (204). (D) SunTag is a repeating protein-peptide array that recruits multiple antibody-fusion proteins. Protein domains, such as transcriptional activating or epigenetic modulating domains, can be recruited by antibody-mediated binding to SunTag, which is fused with dCas (205, 206). (E) RNA aptamers (e.g., MS2, com, PP7) can be fused with sgRNA to create a scaffold RNA (scRNA) that can recruit RNA-binding proteins (RBPs, e.g., MCP, Com, PCP). Fusing each RBP to the effector protein has enabled gene activation, repression, or even simultaneous activation and repression in one cell (207). (F) In the synergistic activation mediator (SAM) system, effectors are recruited by both dCas and scRNA (208). (G) Epigenetic modifying enzymes like P300 and LSD1 can be fused with dCas and alter cells’ epigenomic features. These alterations were locus-specific epigenetic editing, including histone modifications and DNA methylations (205, 209, 210).
FIGURE 4
FIGURE 4
Some strategies to solve CRISPR system limitations. (a) Systems that need dimerization to cleave both strands: 1. dCas-FOK1 2. nCas9. (b) Cas9 variants’ activity can be switched on and off by cell-permeable molecules: (1) Intein-Cas9 is activated by excision of the intein bound to a specific position in Cas9. A cell-permeable small molecule induces this excision (189, 211). (2) Split-Cas9 is a Cas9 molecule that has been split into two fragments, and these two can be dimerized via drug-binding dimerization domains and a cell-permeable drug (212). (3) Degron-Cas9 is formed by binding a destabilizing domain (degron) to Cas9 protein. Previous studies have introduced a different type of degron that can bind to the protein of interest and decrease the stability of that protein in the presence or absence of specific small molecules. Degron domains can also be fused to the RBP-effector complex to regulate its stability and activity of the CRISPR system as a result (, –216).
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