Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells

Wookjae Lee,Duhee Bang,Ji Hyun Lee,Joon Ho Lee,Soyeong Jun

doi:10.1038/s41598-018-30205-2

Wookjae Lee, Duhee Bang + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-30205-2

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Abstract

Mutations within the KRAS oncogene are associated with the proliferation of various cancers. Therapeutic approaches for treating cancers with such mutations have focused on targeting the downstream protein effectors of KRAS. However, to date, no approved treatment has targeted the mutated KRAS oncogene directly. Presently, we used the selectivity of the CRISPR/Cas9 system to directly target mutated KRAS alleles. We designed single-guide RNAs (sgRNAs) to target two specific single-nucleotide missense mutations on KRAS codon-12 located in the seed region adjacent to a protospacer adjacent motif (PAM). Lentiviral transduction of Cas9 and the sgRNAs into cancer cells with respective KRAS mutations resulted in high frequency of indels in the seed region. Indel-associated disruption of the mutant KRAS alleles correlated with reduced viability of the cancer cells. The results indicate that CRISPR-Cas9-mediated genome editing can potentially be used for the treatment of cancer patients, specifically those with oncogenic KRAS mutations.

Highlights

Combined global effort has identified over 600 cancer-inducing somatic mutations that have been catalogued online (COSMIC v82 database)
G > T changes the encoded glycine to a valine (G12V; Fig. 1a). This mutated target nucleotide is located within the seed region adjacent to the protospacer adjacent motif (PAM) sequence, and was chosen to be targeted by clustered regularly interspaced short palindromic repeats (CRISPR)/ Cas[9]
The present study demonstrated a novel method for inhibiting the growth of cancer cell lines with oncogenic KRAS by targeting the mutated gene using CRISPR/Cas[9]

Summary

Introduction

Combined global effort has identified over 600 cancer-inducing somatic mutations that have been catalogued online (COSMIC v82 database). Due to the difficulties of targeting protein directly, a recent paper showed that mutated KRAS gene can be targeted with a synthetic alkylating agent of pyrrole–imidazole polyamide indole-seco-CBI conjugate[18]. Correction of DSBs via error-prone NHEJ commonly results in random insertions or deletions (indels), potentially rendering the target gene nonfunctional. Such disruption of target genes may be beneficial for targeting oncogenes. We developed a method to impair cancer cell proliferation by directly targeting two specific mutations at codon-12 of the KRAS oncogene. We reasoned that the CRISPR/Cas[9] system would selectively target and disrupt the oncogenic alleles, leading to inhibition of cancer cell growth. KRAS mutant-specific CRISPR/Cas[9] selectively inhibited the growth of various cancer cell lines with KRAS mutations in vitro

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