Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
Silvana Konermann,Mark D. Brigham,Alexandro E. Trevino,Julia Joung,Omar O. Abudayyeh,Clea Barcena,Patrick D. Hsu,Naomi Habib,Jonathan S. Gootenberg,Hiroshi Nishimasu,Osamu Nureki,Feng Zhang +11 more
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TLDR
Structural-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci is described and the potential of Cas9-based activators as a powerful genetic perturbation technology is demonstrated.Abstract:
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.read more
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Crystal structure of Cas9 in complex with guide RNA and target DNA
Takashi Yamano,Hiroshi Nishimasu,Hiroshi Nishimasu,Bernd Zetsche,Hisato Hirano,Ian Slaymaker,Yinqing Li,Iana Fedorova,Takanori Nakane,Kira S. Makarova,Eugene V. Koonin,Ryuichiro Ishitani,Feng Zhang,Osamu Nureki +13 more
TL;DR: In this article, the crystal structure of the CRISPR-associated endonuclease Cas9 in complex with sgRNA and its target DNA at 2.5-A resolution was reported.
Journal ArticleDOI
C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector
Omar O. Abudayyeh,Jonathan S. Gootenberg,Silvana Konermann,Silvana Konermann,Silvana Konermann,Julia Joung,Julia Joung,Julia Joung,Ian Slaymaker,Ian Slaymaker,Ian Slaymaker,David Benjamin Turitz Cox,Sergey Shmakov,Sergey Shmakov,Kira S. Makarova,Ekaterina Semenova,Leonid Minakhin,Konstantin Severinov,Konstantin Severinov,Konstantin Severinov,Aviv Regev,Aviv Regev,Eric S. Lander,Eric S. Lander,Eric S. Lander,Eugene V. Koonin,Feng Zhang +26 more
TL;DR: LshC2c2 is a RNA-guided RNase which requires the activity of its two HEPN domains, suggesting previously unidentified mechanisms of RNA targeting and degradation by CRISPR systems.
Journal ArticleDOI
Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers
Isaac B. Hilton,Anthony D'Ippolito,Christopher M. Vockley,Pratiksha I. Thakore,Gregory E. Crawford,Timothy E. Reddy,Charles A. Gersbach +6 more
TL;DR: A programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyl transferase p300 is described, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers.
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