title: Multiplex genome engineering using CRISPR/Cas systems.
abstract: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
authors.affiliation: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA.
authors.forename: Le
authors.initials: L
authors.lastname: Cong
authors.forename: F Ann
authors.initials: FA
authors.lastname: Ran
authors.forename: David
authors.initials: D
authors.lastname: Cox
authors.forename: Shuailiang
authors.initials: S
authors.lastname: Lin
authors.forename: Robert
authors.initials: R
authors.lastname: Barretto
authors.forename: Naomi
authors.initials: N
authors.lastname: Habib
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Hsu
authors.forename: Xuebing
authors.initials: X
authors.lastname: Wu
authors.forename: Wenyan
authors.initials: W
authors.lastname: Jiang
authors.forename: Luciano A
authors.initials: LA
authors.lastname: Marraffini
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D004247:DNA / Q000737:chemistry / Q000235:genetics; D053837:DNA Cleavage; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D016678:Genome / Q000235:genetics*; D006801:Humans; D055029:Inverted Repeat Sequences / Q000235:genetics*; D051379:Mice; D046228:Microarray Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012313:RNA / Q000737:chemistry / Q000235:genetics; D059767:Recombinational DNA Repair; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics
chemical_list: D012313:RNA; D004247:DNA