Harnessing CRISPR-Cas9 immunity for genetic engineering

Abstract

CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.

Figure 1. Cas9-based genetic applications

(A) Wild-type Cas9 loaded with a single guide RNA (sgRNA) generates dsDNA breaks that can be used to introduce target mutations. Chromosomal breaks can be repaired by non-homologous end joining (NHEJ), creating indels that introduce knock-out frameshift mutations. If a sequence homologous to the Cas9 target is provided (the editing template; either linear dsDNA or a short oligonucleotide), the break can be repaired by homologous recombination. In this case, site-specific mutations in the editing template can also be incorporated in the genome. (B) A catalytically dead Cas9 (dCas9, containing mutations in both the RuvC and HNH actives sites) can be used as an RNA-guided DNA binding protein that can repress both transcription initiation when bound to promoter sequences or transcription elongation when bound to the template strand within an open reading frame. Arrows indicate transcription start sites.