Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene


Specifically targeting genomic rearrangements and mutations in tumor cells remains an elusive goal in cancer therapy. Here, we used Cas9-based genome editing to introduce the gene encoding the prodrug-converting enzyme herpes simplex virus type 1 thymidine kinase (HSV1-tk) into the genomes of cancer cells carrying unique sequences resulting from genome rearrangements. Specifically, we targeted the breakpoints of TMEM135–CCDC67 and MAN2A1–FER fusions in human prostate cancer or hepatocellular carcinoma cells in vitro and in mouse xenografts. We designed one adenovirus to deliver the nickase Cas9D10A and guide RNAs targeting the breakpoint sequences, and another to deliver an EGFP-HSV1-tk construct flanked by sequences homologous to those surrounding the breakpoint. Infection with both viruses resulted in breakpoint-dependent expression of EGFP-tk and ganciclovir-mediated apoptosis. When mouse xenografts were treated with adenoviruses and ganciclovir, all animals showed decreased tumor burden and no mortality during the study. Thus, Cas9-mediated suicide-gene insertion may be a viable genotype-specific cancer therapy.

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Figure 1: Schematic of strategy to introduce EGFP-tk into the breakpoint of the TMEM135–CCDC67 fusion gene.
Figure 2: EGFP-tk integration and expression in cells expressing the TMEM135–CCDC67-fusion breakpoint transcript.
Figure 3: Treatment with the nucleotide analog ganciclovir kills cancer cells expressing EGFP-tk.
Figure 4: Treatment with ganciclovir induces partial remission of xenografted prostate cancers in SCID mice.
Figure 5: Genome therapy targeting at the MAN2A1–FER breakpoint.


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We thank S. Zheng for technical support. This work was supported by grants from the National Cancer Institute to JHL (RO1 CA098249 to J.-H.L.), the Department of Defense (W81XWH-16-1-0364) to J.-H.L. and the University of Pittsburgh Cancer Institute to J.-H.L., G.K.M. and J.B.N.

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J.-H.L. and Y.P.Y. conceived the concept of the project and devised the research strategy. Z.-H.C. and Z.-H.Z. performed most experiments, S.M. provided materials, G.K.M. and J.B.N. provided expertise and advice on the biology of and therapies for liver cancer and prostate cancer. S.L. and G.T. performed biostatistics and bioinformatics analyses.

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Correspondence to Jian-Hua Luo.

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The authors declare no competing financial interests.

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Chen, Z., Yu, Y., Zuo, Z. et al. Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene. Nat Biotechnol 35, 543–550 (2017).

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