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Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
npg 2013 Nature America, Inc. All rights reserved.
Kevin M Esvelt1,4, Prashant Mali2,4, Jonathan L Braff1, Mark Moosburner2, Stephanie J Yaung13 & George M Church1,2
a cornucopia of Cas9-mediated transcriptional activators, repressors, fluorescent protein labels, chromosome tethers and numerous other tools.
Although the Cas9 protein from S. pyogenes can mediate one activity at many different target sites, it cannot concurrently mediate a different activity at other targets. For example, a cell engineered with a Cas9 activator cannot undergo genome editing using a Cas9 nuclease without it also cutting the sites being targeted by the activator. Simultaneously employing multiple RNA-guided activities within a single cell will require methods of independently targeting each activity to its own set of target sites. To establish this level of control23,24, we developed methods enabling the characterization of orthogonal Cas9 proteins for multiplexed RNA-guided transcriptional activation, repression and gene editing.
RESULTSSelecting putatively orthogonal Cas9 proteinsCas9 RNA binding and sgRNA specificity is primarily determined by the ~36-bp repeat sequence in pre-crRNA. We began by examining known Cas9 genes for highly divergent repeats in their adjacent CRISPR loci. We chose the genes encoding the well-studied Cas9 protein from S. pyogenes (SP), the smaller Cas9 proteins from Streptococcus thermophilus CRISPR1 and N. meningitidis (ST1 and NM, respectively) and the large Cas9 protein from Treponema denticola (TD). The CRISPR loci associated with these genes harbor repeats that differ by at least 13 nucleotides from one another (Fig. 1a).
Protospacer-adjacentmotif characterization
Known Cas9 proteins will target only dsDNA sequences flanked by a 3 PAM sequence specific to the Cas9 of interest. Of the four Cas9 variants, only SP has an experimentally characterized PAM, whereas the ST1 PAM and, very recently, the NM PAM were deduced bioinformatically. SP is thought to be the most readily targetable of these owing to its short PAM of NGG10; ST1 and NM targeting are constrained by PAMs of NNAGAAW and NNNNGATT, respectively25,26. We hypothesized that, because
1Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts, USA. 2Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. 3Program in Medical Engineering & Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 4These authors contributed...