PAM-flexible genome editing with an engineered chimeric Cas9

Lin Zhao, Sabrina R. T. Koseki, Rachel A. Silverstein, Nadia Amrani, Christina Peng, Christian Kramme, Natasha Savic, Martin Pacesa, Tomás C. Rodríguez, Teodora Stan, Emma Tysinger, Lauren Hong, Vivian Yudistyra, Manvitha R. Ponnapati, Joseph M. Jacobson, George M. Church, Noah Jakimo, Ray Truant, Martin Jinek, Benjamin P. Kleinstiver, Erik J. Sontheimer and Pranam Chatterjee ()
Additional contact information
Lin Zhao: Duke University
Sabrina R. T. Koseki: Duke University
Rachel A. Silverstein: Center for Genomic Medicine, Massachusetts General Hospital
Nadia Amrani: University of Massachusetts Medical School
Christina Peng: McMaster University
Christian Kramme: Harvard University
Natasha Savic: McMaster University
Martin Pacesa: University of Zurich
Tomás C. Rodríguez: University of Massachusetts Medical School
Teodora Stan: Duke University
Emma Tysinger: Duke University
Lauren Hong: Duke University
Vivian Yudistyra: Duke University
Manvitha R. Ponnapati: Massachusetts Institute of Technology
Joseph M. Jacobson: Massachusetts Institute of Technology
George M. Church: Harvard University
Noah Jakimo: Massachusetts Institute of Technology
Ray Truant: McMaster University
Martin Jinek: University of Zurich
Benjamin P. Kleinstiver: Center for Genomic Medicine, Massachusetts General Hospital
Erik J. Sontheimer: University of Massachusetts Medical School
Pranam Chatterjee: Duke University

Nature Communications, 2023, vol. 14, issue 1, 1-8

Abstract: Abstract 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.

Date: 2023
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DOI: 10.1038/s41467-023-41829-y

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