Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing

By Kevin M. Esvelt, P. Mali, Jonathan L. Braff, Mark Moosburner, S. Yaung, George M. Church. Nature Methods, 2013

674 citations recorded by the source corpus.

The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/552a0ff8377e3970d24ab94440565d0dcfdb8ed4

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Citations in the corpus

The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “In this context, another peculiar feature of the CRISPR/Cas9 system is the ability to use orthogonal Cas9 proteins to separately and simultaneously carry out genome editing and gene regulation in the same cell (Esvelt et al., 2013).”
Genetic circuit design automation (2016, positive): “Within this paradigm, the regulators performing the gate biochemistry could be transcrip-tionfactors( 21 , 63 ),RNA-basedregulation( 64 – 66 ), protein-protein interactions ( 19 , 67 ), CRISPR/ Cas-based regulation ( 68 – 71 ), or recombinases ( 25 , 26 , 72 ).”
The new frontier of genome engineering with CRISPR-Cas9 (2014, positive): “The reported collection of Cas9 orthologs constitutes a large source of CRISPR-Cas9 systems for multiplex gene targeting, and several orthologous CRISPR-Cas9 systems have already been applied successfully for genome editing in human cells [Neisseria meningitidis (73, 74), S.”
Development and Applications of CRISPR-Cas9 for Genome Engineering (2014, positive): “NmCas9 and SpCas9, for example, can be employed for independent transcriptional repression and nuclease activity (Esvelt et al., 2013).”
Identifying and visualizing functional PAM diversity across CRISPR-Cas systems (2016, methodology): “thermophilusCRISPR1Cas9 recognizesmultiple sequences that deviate from the consensus NNAGAAW PAM (where W is A or T) (Esvelt et al., 2013; Horvath et al., 2008; Kleinstiver et al., 2015).”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “, Sa Cas9 (69), St1 Cas9, and Nm Cas9 (71)].”