PAM-flexible genome editing with an engineered chimeric Cas9.

• DOI: 10.5281/zenodo.8274440
• Publication Date: 2023-10-04
• Authors: Zhao, Lin; Koseki, Sabrina R T; Silverstein, Rach...; Amrani, Nadia; Peng, Christina; Kramme, Christian; Savic, Natasha; Pacesa, Martin; Rodríguez, Tomás C; Stan, Teodora; Tysinger, Emma; Hong, Lauren; Yudistyra, Vivian; Ponnapati, Manvit...; Jacobson, Joseph M; Church, George M; Jakimo, Noah; Truant, Ray; Jinek, Martin; Kleinstiver, Benj...; Sontheimer, Erik J; Chatterjee, Pranam
• Keywords: CRISPR-Cas9 genome editing; Gene Editing; Genome; CRISPR, PAM, Genome Editing; Science; Q; 610 Medicine & health; 1600 General Chemistry; 3100 General Physics and Astronomy; Article; Targeted gene repair; 1300 General Biochemistry, Genetics and Molecular Biology; CRISPR-Associated Protein 9; 10019 Department of Biochemistry; 570 Life sciences; biology; CRISPR-Cas Systems

AbstractCRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.