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Nat Biotechnol. 2013 Sep;31(9):833-8. doi: 10.1038/nbt.2675. Epub 2013 Aug 1.

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.

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1
1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2].

Abstract

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. 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. 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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PMID:
23907171
PMCID:
PMC3818127
DOI:
10.1038/nbt.2675
[Indexed for MEDLINE]
Free PMC Article

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