title: DNA-binding-domain fusions enhance the targeting range and precision of Cas9.
abstract: The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells. Here we show that by fusing a programmable DNA-binding domain (pDBD) to Cas9 and attenuating Cas9's inherent DNA-binding affinity, we were able to produce a Cas9-pDBD chimera with dramatically improved precision and an increased targeting range. Because the specificity and affinity of this framework can be easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus.
authors.affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Mehmet Fatih
authors.initials: MF
authors.lastname: Bolukbasi
authors.affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Ankit
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Oikemus
authors.affiliation: Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Derr
authors.affiliation: Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.|Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Garber
authors.affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Brodsky
authors.affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.|Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.|Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Lihua Julie
authors.initials: LJ
authors.lastname: Zhu
authors.affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Scot A
authors.initials: SA
authors.lastname: Wolfe
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D004268:DNA-Binding Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems