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

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 prote...

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Bibliographic Details
Published in:Nature biotechnology 2013-09, Vol.31 (9), p.833-838
Main Authors: Mali, Prashant, Aach, John, Stranges, P Benjamin, Esvelt, Kevin M, Moosburner, Mark, Kosuri, Sriram, Yang, Luhan, Church, George M
Format: Article
Language:English
Subjects:
Base Sequence
CRISPR-Associated Proteins - genetics
Deoxyribonuclease I - genetics
Enzymes
Gene expression
Genetic aspects
Genetic engineering
Genetic Engineering - methods
Genetic transcription
Genomics
HEK293 Cells
Humans
Models, Genetic
Molecular Sequence Data
Ribonucleic acid
RNA
RNA sequencing
RNA, Guide - genetics
Trans-Activators - genetics
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Summary: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.
ISSN:1087-0156
1546-1696
DOI:10.1038/nbt.2675