title: Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.
abstract: Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2].
authors.forename: John G
authors.initials: JG
authors.lastname: Doench
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2].
authors.forename: Ella
authors.initials: E
authors.lastname: Hartenian
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Graham
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Brigham and Women's Hospital, Harvard Medical School, Department of Medicine, Division of Hematology, Boston, Massachusetts, USA.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Tothova
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Mudra
authors.initials: M
authors.lastname: Hegde
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Smith
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Meagan
authors.initials: M
authors.lastname: Sullender
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Brigham and Women's Hospital, Harvard Medical School, Department of Medicine, Division of Hematology, Boston, Massachusetts, USA.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Ebert
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA. [3] Center for Computational and Integrative Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Ramnik J
authors.initials: RJ
authors.lastname: Xavier
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Root
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005434:Flow Cytometry; D020868:Gene Silencing; D018390:Gene Targeting; D006801:Humans; D051379:Mice; D012313:RNA / Q000235:genetics*; D013297:Streptococcus pyogenes / Q000235:genetics
chemical_list: D012313:RNA