Because of its high efﬁciency and accuracy, the CRISPR-Cas9 gene editing technique has been widely used in cancer therapeutic explorations. Several studies used CRISPR-Cas9 to directly target cancer cell genomic DNA in cellular and animal cancer models which have shown therapeutic potential in expanding the anticancer protocols. Moreover, CRISPR-Cas9 can also be employed to ﬁght oncogenic infections, explore anticancer drugs, and engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. And CRISPR-Cas9 screens are a powerful functional genomics tool to discover novel targets for cancer therapy. In summary, CRISPR-Cas9 promises to accelerate cancer research by providing an eﬃcient technology to dissect mechanisms of tumorigenesis, identify targets for drug development.
The application of CRISPR-Cas9 to several aspects of cancer biology, ranging from basic research to clinical and translational applications, offers numerous exciting opportunities for better understanding and potentially treating this devastating disease. The rapid development of the CRISPR-Cas9-mediated gene editing tool has revolutionized the gene therapy area, which not only holds extensive application potential for therapeutic manipulations of cancer genomes, but also can be used to ﬁght oncogenic infections, modulate gene expression, and explore anti-cancer drugs. Besides targeting cancer cell genomes directly, the CRISPR-Cas9 system can also be applied for precise engineering of immune cells and oncolytic viruses for cancer immunotherapeutic applications.
Given that cancer accumulates multiple genetic alterations and that CRISPR-Cas9 can be harnessed for rapidly and precisely engineering both loss-of-function (LOF) and gain-of-function (GOF) mutations in tumor suppressor genes, oncogenes and other modulators of cellular transformation or drug response, it is reasonable to predict that CRISPR-Cas9 can be used to correct gene aberrations that drive cancer pathogenesis and development or to target knockout necessities for cancer cell survival and chemo-resistant genes. Mutated oncogenes and tumor suppressor genes in cancer cells are attractive therapeutic targets in cancer. The CRISPR-Cas9 system can also be utilized to trigger two distant DSBs in the same or different chromosomes, leading to inversion, deletion or translocation of the target or translocation of the target sequences, respectively.
dCas9-effector fusions also have potential applications in cancer biology. The ability of Cas9 to bind in a specific RNA-dependent fashion can be uncoupled from its nuclease activity by mutating its HNH and RuvC-like catalytic domains. This catalytically inactive form of Cas9 (dCas9), retains its RNA-guided DNA binding activity without any detectable DNA endonuclease activity. Researchs have demonstrated the power of dCas9-effector fusions for reversible transcriptional repression or activation of endogenous coding and non-coding genes. In addition, the use of scaffold RNAs that encode both targeting and effector-recruitment functions can be utilized for simultaneous multiplex gene repression and activation within a single cell. The ability to multiplex the CRISPR-Cas9 system offers opportunity to investigate combinatorial vulnerabilities in cancer cells.
The flexibility of the CRISPR-Cas9 technology has been exploited for carrying out high-throughput CRISPR screens using the Cas9 nuclease and dCas9-effectors for the systematic identification of genes involved in a variety of biological phenotypes. Moreover, combined with genome sequencing and resistance selection, CRISPR-Cas9 systems have also been widely adopted to conduct genome-scale screens for drug resistance genes. CRISPR-Cas9 is a tool to rapidly and systematically identify drug targets and resistance mechanisms, which would be beneﬁcial for anti-cancer drug development. Compared to RNA interference screens, CRISPR-Cas9-mediated screening showed a higher validation rate and reagent consistency. In the future, pooled CRISPR screens will provide a comprehensive set of essential genes across most cancer cell lines.
Although the CRISPR-Cas9 system has attracted tremendous attention for its potential in cancer therapy, many challenges remain to be addressed to fully realize the clinical applications. One primary challenge is the potential off-target effects. When applied for therapeutic purposes, even very low off-target editing can be detrimental. Thus, the off-target effects of CRISPR-Cas9 must be accurately identiﬁed and controlled, and avoided as much as possible to alleviate or prevent damage to normal cell genomes. The off-target DSBs could lead to small indels or large-scale genomic alternations including large deletions, inversions, and translocations at nontarget sites. The large-scale off-target genomic alternations can be readily detected through modalities. However, small off-target mutations may be difﬁcult to analyze.
Another major challenge is the efﬁcient delivery of CRISPR-Cas9 components into target tissues, which is particularly important for cancer therapy. Another challenge in applying CRISPR-Cas9 in cancer patients may be the editing efﬁciency. CRISPR-Cas9-mediated genome manipulation for cancer therapy requires an extremely high level of editing efﬁciency because the unmodiﬁed cells proliferate more rapidly than the modiﬁed ones, as a result, nullifying the treatment effect quickly and leading to relapse. The editing efﬁciency of CRISPR-Cas9 lies in the careful selection of target sites, potent Cas9, efﬁcient delivery vectors, and powerful sgRNA; hence, further insights into these issues will lead to higher editing efﬁciency.
Although signiﬁcant progress has been made in overcoming these aforementioned challenges for clinically applying CRISPR-Cas9, it is still a long road to fully realize the use of CRISPR-Cas9-based gene editing as a therapeutic strategy to target cancer genes in human patients. In principle, the therapeutic beneﬁt and the predictable risk of gene editing should be carefully assessed prior to the clinical application. Anyhow, the development of the CRISPR-Cas9 technology has and will greatly accelerate cancer research in many areas.
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