CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
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 e...
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Published in | Nature biotechnology Vol. 31; no. 9; pp. 833 - 838 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
New York
Nature Publishing Group US
01.09.2013
Nature Publishing Group |
Subjects | |
Online Access | Get full text |
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Summary: | 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
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. 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
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. 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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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 These authors contributed equally to this work |
ISSN: | 1087-0156 1546-1696 |
DOI: | 10.1038/nbt.2675 |