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. 2017 Jul 27;45(13):7897-7908.
doi: 10.1093/nar/gkx490.

Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression

Taeyoung Koo  1   2 , A-Rum Yoon  3 , Hee-Yeon Cho  1 , Sangsu Bae  4 , Chae-Ok Yun  3 , Jin-Soo Kim  1   2   5
Affiliations

Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression

Taeyoung Koo et al. Nucleic Acids Res. .

Abstract

Approximately 15% of non-small cell lung cancer cases are associated with a mutation in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. With the goal of treating mutated EGFR-mediated lung cancer, we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system to discriminate between the oncogenic mutant and wild-type EGFR alleles and eliminate the carcinogenic mutant EGFR allele with high accuracy. We _targeted an EGFR oncogene harboring a single-nucleotide missense mutation (CTG > CGG) that generates a protospacer-adjacent motif sequence recognized by the CRISPR/Cas9 derived from Streptococcus pyogenes. Co-delivery of Cas9 and an EGFR mutation-specific single-guide RNA via adenovirus resulted in precise disruption at the oncogenic mutation site with high specificity. Furthermore, this CRISPR/Cas9-mediated mutant allele disruption led to significantly enhanced cancer cell killing and reduced tumor size in a xenograft mouse model of human lung cancer. Taken together, these results indicate that _targeting an oncogenic mutation using CRISPR/Cas9 offers a powerful surgical strategy to disrupt oncogenic mutations to treat cancers; similar strategies could be used to treat other mutation-associated diseases.

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Figures

Figure 1.
Figure 1.
Oncogenic mutant-specific Cas9. (A) A PAM sequence (red) is generated by a single nucleotide missense mutation in the EGFR gene. The corresponding wild-type sequence is shown in green. The sgRNA _target sequence is shown in blue. (B) Diagram of selective cancer cell killing by cleavage of the oncogenic mutant allele 3-bp upstream of the PAM. (C) Mutation frequencies at the EGFR _target site in H1975 cells or A549 cells co-transfected with Ad/sgEGFR and Ad/Cas9 48 h post-transfection. Mismatched nucleotides are shown in yellow and PAM sequences in red. The sgRNA _target sequence is shown in blue. The column on the right indicates the number of inserted or deleted bases. Bars represent the mean ± S.E.M (n = 3). **P < 0.01, ns: not significant.
Figure 2.
Figure 2.
Specific excision of the oncogenic EGFR allele induces cytopathic effect. (A) Representative diagram of E1-deleted adenovirus type 5 encoding either SpCas9 or sgRNA _targeting the EGFR gene. (B) Mutation frequencies at the EGFR _target site in H1975 and A549 cells were examined via _targeted deep sequencing day 1, 2 and 5 after co-transduction of Ad/sgEGFR and Ad/Cas9 with MOIs of 10, 20 and 50. (C) EGFR mRNA levels, normalized with an 18S rRNA internal control, were determined day 2 after co-infection of H1975 cells with Ad/sgEGFR and Ad/Cas9. (D) CRISPR/Cas9-mediated cell killing efficacy in H1975 or A549 cells. Cells were treated with Ad/sgEGFR, Ad/Cas9, or Ad/sgEGFR + Ad/Cas9; day 2 post-treatment, an MTT assay was performed. Bars represent the mean ± S.E.M (n = 3). * (P < 0.05), ** (P < 0.01), *** (P < 0.001), ns: not significant.
Figure 3.
Figure 3.
Selective oncogene disruption using Ad/sgEGFR and Ad/Cas9 in vivo. Indels at the wild-type (gray bar) and mutant EGFR alleles (blue bar) in (A) H1975 or (B) A549 tumor xenografts co-injected with Ad/sgEGFR and Ad/Cas9 at day 7, 9, 11 and 31 after the first injection. Tumor-bearing mice were given intratumoral injections of PBS (mock) or 5 × 1010 Ad VPs (Ad/Cas9 + Ad/empty vector or Ad/sgEGFR + Ad/Cas9) on days 1, 3 and 5. Values represent the mean ± S.E.M. (n= 3 to 4). **P < 0.01. ns: not significant. (C) No off-_target indels were detectably induced at eight homologous sites that differed from the on-_target sites by up to 3 nt in the human genome. Mismatched nucleotides are shown in blue and PAM sequences in red, On; on-_target site, OT; off-_target site. Red arrow indicates cleavage position within the 19-bp _target sequences. Error bar indicates S.E.M. (n = 3 to 4).
Figure 4.
Figure 4.
Antitumor effect and survival benefit of adenoviral delivery of CRISPR/Cas9 to tumor xenograft models. (A) H1975 tumor-bearing mice were given intratumoral injections of PBS or 5 × 1010 VPs (Ad/Cas9 + Ad/empty vector or Ad/sgEGFR and Ad/Cas9) on days 1, 3 and 5. Tumor growth was monitored every other day until 31 days post injection. Values represent the mean ± S.E.M. for eight animals per group. ***P < 0.001 compared with the PBS and Ad/Cas9 treated groups. The percentage of surviving mice was determined by monitoring tumor growth-related events (tumor size < 800 mm3) over a time period. (B) A549 tumor-bearing mice were given intratumoral injections of PBS or 5 × 1010 Ad VPs (Ad/sgEGFR and Ad/Cas9) on days 1, 3 and 5. Tumor growth was monitored every other day until the end of the study. Bar represent the mean ± S.E.M. for eight animals per group. NS; not significant compared with the PBS treated groups. The percentage of surviving mice was determined by monitoring tumor growth-related events (tumor size < 800 mm3) over a time period. (C) PBS, Ad/Cas9 + Ad/empty vector or Ad/sgEGFR and Ad/Cas9 was injected on days 1, 3 and 5 into established H1975 tumors in nude mice. Tumors were harvested on day 7 for histological analysis. H & E staining and immunohistochemical staining of PCNA and EGFR were performed on tumor sections from each group of mice.
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