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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. .

Erratum in

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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