Abstract
Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.
Highlights
Methods for introducing site-specific double-strand DNA breaks (DSBs) in genomic DNA have transformed our ability to engineer eukaryotic organisms by initiating DNA repair pathways that lead to targeted genetic re-programming
These results show that RNA-programmed genome editing is a straightforward strategy for introducing site-specific genetic changes in human cells, and the ease with which it can programmed means that it is likely to become competitive with existing approaches based on zinc finger nucleases and transcription activator-like effector nucleases, and could lead to a new generation of experiments in the field of genome engineering for humans and other species with complex genomes
We show here that Cas9 can be expressed and localized to the nucleus of human cells, and that it assembles with sgRNA in vivo.These complexes can generate double stranded breaks and stimulate non-homologous end joining (NHEJ) repair in genomic DNA at a site complementary to the sgRNA sequence, an activity that requires both Cas9 and the sgRNA
Summary
Methods for introducing site-specific double-strand DNA (dsDNA) breaks (DSBs) in genomic DNA have transformed our ability to engineer eukaryotic organisms by initiating DNA repair pathways that lead to targeted genetic re-programming. Using sgRNA, Cas can be programmed to cleave double-stranded DNA at any site defined by the guide RNA sequence and including a GG protospacer-adjacent (PAM) motif (Sapranauskas et al, 2011; Jinek et al, 2012). These findings suggested the exciting possibility that Cas9:sgRNA complexes might constitute a simple and versatile RNA-directed system for generating DSBs that could facilitate site-specific
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