Rationale: KRAS 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. Methods: Two CRISPR 
systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, 
were developed to deplete the KRAS G12S mutant allele or repress its 
transcription, respectively, with the goal of treating KRAS-driven cancers. 
Results: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele 
blocked the expression of the mutant KRAS gene, leading to an inhibition of 
cancer cell proliferation. Local adenoviral injections using SpCas9 and 
dCas9-KRAB systems suppressed tumor growth in vivo. 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 
KRAS 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. Conclusion: We 
successfully developed both gene-depletion and transcription-suppressing systems 
to specifically target an oncogenic KRAS 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.