Modification of the human KRAS gene using CRISPR/Cas9 system
Fok, Ezio Tony
The genome is comprised of a simple quaternary code that serves as the “software” which governs all the complex processes of life. The successful manipulation of this code holds enormous potential for applications involving genome engineering. However, the tools needed to navigate the complex landscape of the genome and efficiently and precisely introduce engineered modifications are lacking. The clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system found in prokaryotes functions to recognise and silence foreign pathogenic DNA by double-stranded break (DSB) DNA digestion. The type II CRISPR system of Streptococcus pyogenes has recently been reconstituted to function in mammalian cells as a highly programmable and efficient gene-targeting nuclease platform. Through the heterologous expression of the CRISPR associated (Cas) 9 endonuclease and a small single-guide RNA (sgRNA) molecule, specific gene sequences can be targeted for a DNA DSB. The repair of this targeted DNA damage can be exploited to mutate gene sequences or reconstitute break sites according to a homologous repair template for precise gene modifications. In this study, the capabilities of the CRISPR/ Cas9 system for genome editing was tested by using it to precisely mutate the KRAS proto-oncogene. A panel of five KRAS targeting sgRNAs were designed around a common G>T mutation in codon 12 and characterised. The ability of the CRISPR/ Cas9 system to stimulate DSBs in this region was assessed using the Surveyor Assay, which is able to detect the non-homologous end joining repair of these breaks. A maximum cleavage activity of 6.01 and 2.74% was detected, upstream and downstream of the mutation site, respectively. Simultaneous cleavage by these two sgRNAs was able to successfully introduce a locus-specific micro-deletion. The homology directed repair of these DSBs according to a 90-mer single-stranded oligodeoxynucleotide repair template was shown with the RFLP assay. Analysis of these results implicated guide sequence composition and DSB repair pathway bias as potential factors which may affect the efficiency of desired gene-editing outcomes. These characterised sgRNAs were then applied to generate selectable G>T KRAS mutants. A “dual-cut” strategy, which was designed to overcome gene conversion limitations was employed, and the outcomes were measured with qPCR. The results show that 0.123% of transfected cells were successful recombinants, demonstrating that the use of a “dual-cut” strategy with the CRISPR/Cas9 system was functional and efficient for the generation of knock-in mutants. The CRISPR/Cas9 system has proven to be efficient and robust in modifying the human KRAS locus in various ways. With its modularity and simplicity, CRISPR/Cas9 is a powerful tool that will allow for the modification and interrogation of gene function.
A dissertation Submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Health Sciences at the University of the Witwatersrand, Johannesburg, South Africa. November 2014