Guo, Bin2021-09-152021-09-15May 20212021-05May 2021Portions of this document appear in: Liu, Jingwen, Meiyan Sun, Kwang Bog Cho, Xiang Gao, and Bin Guo. "A CRISPR-Cas9 repressor for epigenetic silencing of KRAS." Pharmacological Research 164 (2021): 105304.https://hdl.handle.net/10657/8252KRAS 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.application/pdfengThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).KRAS mutations, CRISPR, HDAC1, Ribonucleoprotein, Epigenetics2021-09-15Thesisborn digital