title: Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.
abstract: Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.
authors.affiliation: MilliporeSigma, 2909 Laclede Avenue, Saint Louis, Missouri 63103, USA.|A Business of Merck KGaA, 64293 Darmstadt, Germany.
authors.forename: Fuqiang
authors.initials: F
authors.lastname: Chen
authors.affiliation: MilliporeSigma, 2909 Laclede Avenue, Saint Louis, Missouri 63103, USA.|A Business of Merck KGaA, 64293 Darmstadt, Germany.
authors.forename: Xiao
authors.initials: X
authors.lastname: Ding
authors.affiliation: MilliporeSigma, 2909 Laclede Avenue, Saint Louis, Missouri 63103, USA.|A Business of Merck KGaA, 64293 Darmstadt, Germany.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Feng
authors.affiliation: MilliporeSigma, 2909 Laclede Avenue, Saint Louis, Missouri 63103, USA.|A Business of Merck KGaA, 64293 Darmstadt, Germany.
authors.forename: Timothy
authors.initials: T
authors.lastname: Seebeck
authors.affiliation: MilliporeSigma, 2909 Laclede Avenue, Saint Louis, Missouri 63103, USA.|A Business of Merck KGaA, 64293 Darmstadt, Germany.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: MilliporeSigma, 2909 Laclede Avenue, Saint Louis, Missouri 63103, USA.|A Business of Merck KGaA, 64293 Darmstadt, Germany.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Davis
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053837:DNA Cleavage; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D008957:Models, Genetic
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases