Selective targeting of the oncogenic <i>KRAS</i> G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression

<b>Rationale</b>: <i>KRAS</i> is one of the most frequently mutated oncogenes in cancers. The protein's picomolar affinity for GTP/GDP and smooth protein structure resulting in the absence of known allosteric regulatory sites makes its genomic-level activating mutations a difficult but attractive target. <b>Methods</b>: Two CRISPR systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, were developed to deplete the <i>KRAS</i> G12S mutant allele or repress its transcription, respectively, with the goal of treating <i>KRAS</i>-driven cancers. <b>Results</b>: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele blocked the expression of the mutant <i>KRAS</i> gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems suppressed tumor growth <i>in vivo</i>. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type <i>KRAS</i> tumors did not affect cell proliferation. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, the data showed that our mutant-specific editing strategy could be extended to a reference list of oncogenic mutations with high editing potentials. This pipeline could be applied to analyze the distribution of PAM sequences and survey the best alternative targets for gene editing. <b>Conclusion</b>: We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic <i>KRAS</i> mutant allele that led to significant tumor regression. These findings show the potential of CRISPR-based strategies for the treatment of tumors with driver gene mutations.