Abstract
The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.
Highlights
The development of precise and highly efficient genome editing tools is transforming biological research and expediting biotechnological applications ranging from superior crop production to genomic medicine uses [1]
The dead Cas9 protein (dCas9) protein variant was incapable of cleaving the DNA but retained the ability to be targeted by guide RNAs (gRNAs)
With architectures reminiscent of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) architectures, can be used to generate homodimers on a DNA target sequence defined by the specificities of gRNA sequences on sense and antisense strands and the length of the intervening spacer sequence, (Fig 1A) [7]
Summary
The development of precise and highly efficient genome editing tools is transforming biological research and expediting biotechnological applications ranging from superior crop production to genomic medicine uses [1]. Genome editing tools allow precise alteration of DNA sequences on a single nucleotide level. Such control permits functional characterization of genes and their variants and linking a particular genotype to a particular phenotype. Several genome-engineering approaches have been developed including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [2,3,4,5,6,7]. ZFNs are used in a variety of organisms and PLOS ONE | DOI:10.1371/journal.pone.0133373. ZFNs are used in a variety of organisms and PLOS ONE | DOI:10.1371/journal.pone.0133373 July 30, 2015
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