title: Rational design of a split-Cas9 enzyme complex.
abstract: Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.
authors.affiliation: Department of Molecular and Cell Biology.
authors.forename: Addison V
authors.initials: AV
authors.lastname: Wright
authors.affiliation: Department of Chemistry.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Sternberg
authors.affiliation: Howard Hughes Medical Institute, and.
authors.forename: David W
authors.initials: DW
authors.lastname: Taylor
authors.affiliation: Department of Molecular and Cell Biology.
authors.forename: Brett T
authors.initials: BT
authors.lastname: Staahl
authors.affiliation: Biophysics Graduate Group, University of California, Berkeley, CA 94720; and.
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Bardales
authors.affiliation: Department of Molecular and Cell Biology.
authors.forename: Jack E
authors.initials: JE
authors.lastname: Kornfeld
authors.affiliation: Department of Molecular and Cell Biology, Department of Chemistry, Howard Hughes Medical Institute, and Biophysics Graduate Group, University of California, Berkeley, CA 94720; and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 doudna@berkeley.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D013297:Streptococcus pyogenes / Q000201:enzymology; D014158:Transcription, Genetic
chemical_list: D004247:DNA
keywords: CRISPR-Cas9; genome engineering; split enzyme