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

• Published in: Nature biotechnology Vol. 31; no. 9; pp. 833 - 838
• Main Authors: Mali, Prashant, Aach, John, Stranges, P Benjamin, Esvelt, Kevin M, Moosburner, Mark, Kosuri, Sriram, Yang, Luhan, Church, George M
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.09.2013; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/1513/1967; Agriculture; Base Sequence; Bioinformatics; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; CRISPR-Associated Proteins - genetics; Deoxyribonuclease I - genetics; Enzymes; Gene expression; Genetic aspects; Genetic engineering; Genetic Engineering - methods; Genetic transcription; Genomics; HEK293 Cells; Humans; letter; Life Sciences; Models, Genetic; Molecular Sequence Data; Ribonucleic acid; RNA; RNA sequencing; RNA, Guide - genetics; Trans-Activators - genetics; United States
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt.2675

A screen in human cells defines the targeting specificities of sgRNA:Cas9 and TAL-based transcriptional activators. Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes 1 , 2 , 3 , 4 , 5 , 6 , 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.