title: Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.
abstract: CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: John G
authors.initials: JG
authors.lastname: Doench
authors.affiliation: Microsoft Research New England, Cambridge, Massachusetts, USA.
authors.forename: Nicolo
authors.initials: N
authors.lastname: Fusi
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Meagan
authors.initials: M
authors.lastname: Sullender
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: Emma W
authors.initials: EW
authors.lastname: Vaimberg
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Katherine F
authors.initials: KF
authors.lastname: Donovan
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.|Dana Farber Cancer Institute, Division of Hematologic Malignancies, Boston, Massachusetts, USA.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Tothova
authors.affiliation: Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri, USA.
authors.forename: Craig
authors.initials: C
authors.lastname: Wilen
authors.affiliation: Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Orchard
authors.affiliation: Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri, USA.
authors.forename: Herbert W
authors.initials: HW
authors.lastname: Virgin
authors.affiliation: Microsoft Research New England, Cambridge, Massachusetts, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Listgarten
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*; D045744:Cell Line, Tumor; D004351:Drug Resistance / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006801:Humans; D051379:Mice; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems