Efficient and specific genome editing in human cells using CRISPR/Cas system

Abstract

Programmable nucleases based on the FokI nuclease domain have been used widely for genome targeting in various cells and organisms. Recent identification of a prokaryotic immune system called CRISPR shows that a CRISPR/Cas system has the prospect of being another programmable nuclease for genome targeting in other higher eukaryotes. In this study, I have used a CRISPR/Cas system as a programmable RNA-guided endonucleases (RGENs), which cleave DNA in a targeted manner for genome engineering in human cells. These enzymes can induce site specific double strand breaks (DSBs) in chromosomal DNA and the repair of DSBs gives rise to targeted genome modifications via non-homologous end joining or homologous recombination. In this study, RGENs can induce efficient mutations at frequencies of up to 75% in human K562 cells. Compared to ZFNs or TALENs, the specificity of RGENs can easily be customized by synthesizing new guide-RNA molecules. Furthermore, I investigated off-target effects of RGENs. Off-target cleavage at unwanted loci can cause genetic instability including tumorigenesis or chromosome structural variations. The present study demonstrates that RGENs can efficiently discriminate on-target sites from off-target sites that differ by two bases. Indeed, exome sequencing analysis shows that RGEN-induced mutant clone without off-target mutations can be isolated. These features will make RGENs the most powerful and applicable tool for genome engineering.