title: Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing.
abstract: Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.
authors.affiliation: The Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Oakes
authors.affiliation: Sangamo BioSciences Inc., Richmond, California 94804, USA.
authors.forename: Danny F
authors.initials: DF
authors.lastname: Xia
authors.affiliation: The Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Elizabeth F
authors.initials: EF
authors.lastname: Rowland
authors.affiliation: The Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Denise J
authors.initials: DJ
authors.lastname: Xu
authors.affiliation: Sangamo BioSciences Inc., Richmond, California 94804, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Ankoudinova
authors.affiliation: Sangamo BioSciences Inc., Richmond, California 94804, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Borchardt
authors.affiliation: Sangamo BioSciences Inc., Richmond, California 94804, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Sangamo BioSciences Inc., Richmond, California 94804, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Li
authors.affiliation: Sangamo BioSciences Inc., Richmond, California 94804, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.affiliation: Sangamo BioSciences Inc., Richmond, California 94804, USA.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.affiliation: The Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.|Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.|Institute for Systems Genetics, NYU School of Medicine, New York, New York 10016, USA.|Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, New York 10016, USA.
authors.forename: Marcus B
authors.initials: MB
authors.lastname: Noyes
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D017930:Genes, Reporter; D016678:Genome; D006801:Humans; D019151:Peptide Library; D054390:Receptors, CCR2 / Q000378:metabolism; D019713:Receptors, CCR5 / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D019151:Peptide Library; D054390:Receptors, CCR2; D019713:Receptors, CCR5; D003851:Deoxyribonucleases