Mali, PrashantAach, JohnStranges, P. BenjaminEsvelt, Kevin MichaelMoosburner, MarkKosuri, SriramYang, LuhanChurch, George2014-04-112013Mali, Prashant, John Aach, P. Benjamin Stranges, Kevin M. Esvelt, Mark Moosburner, Sriram Kosuri, Luhan Yang, and George M. Church. 2013. “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.” Nature biotechnology 31 (9): 10.1038/nbt.2675. doi:10.1038/nbt.2675. http://dx.doi.org/10.1038/nbt.2675.1087-0156http://nrs.harvard.edu/urn-3:HUL.InstRepos:12064510Prokaryotic 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 novel 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 (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins 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.en-USJournal Article2014-04-1110.1038/nbt.2675