CAS9 Transcriptional Activators for Target Specificity Screening and Paired Nickases for Cooperative Genome Engineering

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2013 Aug 3

PMID 23907171

Citations 942

Authors

Prashant Mali

John Aach

P Benjamin Stranges

Kevin M Esvelt

Mark Moosburner

Sriram Kosuri

Luhan Yang

George M Church

Affiliations

Soon will be listed here.

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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