Rational design of a split-Cas9 enzyme complex

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 10; pp. 2984 - 2989
• Main Authors: Wright, Addison V., Sternberg, Samuel H., Taylor, David W., Staahl, Brett T., Bardales, Jorge A., Kornfeld, Jack E., Doudna, Jennifer A.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 10.03.2015; National Acad Sciences; National Academy of Sciences, Washington, DC (United States)
• Subjects: BASIC BIOLOGICAL SCIENCES; Biological Sciences; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas9; Deoxyribonucleic acid; DNA; DNA - metabolism; Electrophoretic Mobility Shift Assay; Enzymes; genome engineering; Gram-positive bacteria; Hydrolysis; Molecules; Nucleic Acid Conformation; Polypeptides; Proteins; Ribonucleic acid; RNA; split enzyme; Streptococcus pyogenes - enzymology; Transcription, Genetic; CRISPR-Cas9; genome engineering; split enzyme
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1501698112

Significance Bacteria have evolved clustered regularly interspaced short palindromic repeats (CRISPRs) together with CRISPR-associated (Cas) proteins to defend themselves against viral infection. RNAs derived from the CRISPR locus assemble with Cas proteins into programmable DNA-targeting complexes that destroy DNA molecules complementary to the guide RNA. In type II CRISPR-Cas systems, the Cas9 protein binds and cleaves target DNA sequences at sites complementary to a 20-nt guide RNA sequence. This activity has been harnessed for a wide range of genome-engineering applications. This study explores the structural features that enable Cas9 to bind and cleave target DNAs, and the results suggest a way of regulating Cas9 by physical separation of the catalytic domains from the rest of the protein scaffold. 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.