Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease

• Main Authors: Joung, Julia (Contributor), Zhang, Feng (Contributor), Konermann, Silvana (Contributor), Dahlman, James E. (Author), Abudayyeh, Omar Osama (Contributor), Gootenberg, Jonathan S. (Contributor)
• Other Authors: Harvard University- (Contributor), Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences (Contributor), McGovern Institute for Brain Research at MIT (Contributor)
• Format: Article
• Language: English
• Published: Nature Publishing Group, 2016-05-22T23:41:33Z.
• Subjects: Article
• Online Access: Get fulltext

We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14- to 15-bp target sequences and MS2 binding loops, can activate gene expression using an active Streptococcus pyogenes Cas9 nuclease, without inducing double-stranded breaks. We use these 'dead RNAs' to perform orthogonal gene knockout and transcriptional activation in human cells.
McGovern Institute for Brain Research at MIT (Friends of McGovern Institute Fellowship)
United States. Dept. of Energy (Computational Science Graduate Fellowship)
National Institute of Mental Health (U.S.) (1DP1-MH100706)
Poitras Foundation
Vallee Foundation
Simons Foundation
Paul G. Allen Foundation
New York Stem Cell Foundation