PAM-flexible genome editing with an engineered chimeric Cas9

作者：Lin Zhao, Sabrina Koseki, Rachel A. Silverstein, Nadia Amrani, Christina Peng, Christian Kramme, Natasha Savic, Martin Pačesa, Tomás Rodríguez, Teodora Stan, Emma Tysinger, Lauren Hong, Vivian Yudistyra, Manvitha Ponnapati, Joseph M. Jacobson, George M. Church, Noah Jakimo, Ray Truant, Martin Jínek, Benjamin P. Kleinstiver, Erik J. Sontheimer, Pranam Chatterjee · 发表于：Nature Communications · 年份：2023 · DOI：10.1038/s41467-023-41829-y · 被引用次数：57 · 研究领域：CRISPR and Genetic Engineering、RNA and protein synthesis mechanisms、RNA regulation and disease

CRISPR 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.