Selective targeting of an oncogenic KRAS mutant allele by CRISPR/Cas9 induces efficient tumor regression
Posted 17 Oct 2019
bioRxiv DOI: 10.1101/807578
Posted 17 Oct 2019
Background KRAS is one of the most frequently mutated oncogenes in human cancers, but its activating mutations have remained undruggable due to its picomolar affinity for GTP/GDP and its smooth protein structure resulting in the absence of known allosteric regulatory sites. Results With the goal of treating mutated KRAS -driven cancers, two CRISPR systems, CRISPR-SpCas9 genome-editing system and transcription-regulating system dCas9-KRAB, were developed to directly deplete KRAS mutant allele or to repress its transcription in cancer cells, respectively, through guide RNA specifically targeting the mutant but not wild-type allele. The effect of in vitro proliferation and cell cycle on cancer cells as well as in vivo tumor growth was examined after delivery of Cas9 system. SpCas9 and dCas9-KRAB systems with sgRNA targeting the mutant allele both blocked the expression of mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems both 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 tumor cells did not affect cell proliferation in vitro and in vivo . Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, we showed that our mutant-specific editing strategy could be extended to a list of oncogenic mutations with high editing potentials, and this pipeline can be applied to analyze the distribution of PAM sequence in the genome to survey the best targets for other editing purpose. Conclusions We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic mutant allele of KRAS which led to significant tumor regression. It provides a promising strategy for the treatment of tumors with driver gene mutations. * PDACs : pancreatic ductal adenocarcinomas CRCs : colorectal adenocarcinomas LACs : lung adenocarcinomas CRISPR : Clustered regularly interspaced short palindromic repeats SpCas9 : S.pyogenes CRISPR associated protein 9 EGFR : epidermal growth factor receptor dCas9 : dead Cas9 KRAB : Krüppel associated box PAM : protospacer adjacent motif NGS : next generation sequencing CFA : colony formation assay DSB : double stand break IHC : immunohistochemical WB : western blot SNV : single nucleotide variation S : sense AS : anti-sense SNP : single-nucleotide polymorphism AAV : adeno-associated viral ATCC : American Type Culture Collection DMEM : Dulbecco’s modified Eagle’s medium GAPDH : glyceraldehyde 3-phosphate dehydrogenase H&E : hematoxylin and eosin
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