A CRISPR-Cas9 Repressor for Epigenetic Silencing of KRAS
KRAS is one of the most frequently mutated oncogenes in cancers. KRAS mutations have been found in in nearly 30% of all human cancers. Despite decades of study, no effective approved anti-KRAS therapies are available. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) system has a great potential for treating human genetic disease due to its genome editing efficiency. In this study, we designed a plasmid by introducing the epigenetic repressor transcriptional repressor histone deacetylase 1 (HDAC1) cDNAs into the pcDNA3.1-dCas9 plasmid. The catalytically dead Cas9 (dCas9) functioned as a DNA binding device, while HDAC1 acted as epigenetic repressor. We designed a panel of three CRISPR RNAs (crRNAs) covering 1500-bp range of the KRAS promoter and identified crRNA1 and crRNA2 for silencing K-Ras efficiently. The inhibition of K-Ras significantly inhibited cell growth, suppressed colony formation in soft agar, induced significant cell death, inhibited protein expression of K-Ras downstream targets and reduced cell migration and invasion in HCT-116 colon cancer cells and NCI-H358 lung cancer cells. In addition, we performed rescue experiments using mutated KRAS plasmid to overexpress K-Ras protein in NCI-H358. The results confirmed that the inhibitory effect of dCas9-HDAC1 system in NCI-H358 is through downregulation of K-Ras. The chromatin immunoprecipitation (ChIP) assay confirmed dCas9-HDAC1 modified histone acetylation on the KRAS promoter. Furthermore, we generated recombinant protein dCas9-HDAC1 in insect cells. The fusion protein was able to target the mutant KRAS promoter and suppress KRAS expression through epigenome editing as well. The suppression of K-Ras by the delivery of dCas9-HDAC1 protein system resulted in a significant inhibition of cell growth, induction of cell death and inhibition of colony formation in soft agar. Finally, we investigated RNA nanoparticle decorated exosomes as a delivery system for ribonucleoprotein (RNP) formed by dCas9-HDAC1 protein and gRNA. The RNA nanoparticle decorated exosomes were able to encapsulate RNP. The delivery system downregulated K-Ras level and inhibited cells proliferation in HCT116 cells. In summary, we have developed a novel strategy that combines CRISPR-Cas9 technology with HDAC1 epigenetic silencing to target cancers driven by KRAS mutations.