Journal
NATURE BIOTECHNOLOGY
Volume 31, Issue 9, Pages 833-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.2675
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Funding
- US National Institutes of Health [P50 HG005550]
- Department of Energy [DE-FG02-02ER63445]
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Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes(1-7). Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA: Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors(8,9). Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA: Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA: Cas9 tool and highlight the critical need to engineer improved specificity.
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