Kariko and Weissman

An exciting study published in Nature Communications on October 4, 2023, authored by Zhao, L., Koseki, S.R.T., Silverstein, R.A., et al., delves into overcoming limitations in CRISPR genome editing by introducing SpRYc—a chimeric Cas9 resulting from the fusion of Sc++ and SpRY. Unlike conventional Cas9 enzymes, SpRYc breaks free from the constraints of specific protospacer adjacent motifs (PAMs) by accommodating a 5’-NNN-3’ PAM preference. The chimeric SpRYc, engineered through a strategic grafting of SpRY’s PAM-interacting domain onto Sc++, showcases remarkable PAM flexibility, allowing specific editing of diverse 5’-NNN-3’ PAM targets in human cells.

In the development of SpRYc, substantial substitutions in the PAM-interacting domain (PID) of SpCas9 endowed it with a more flexible 5’-NRN-3’ PAM specificity. In contrast, ScCas9 and Sc + + leverage positive-charged, flexible loop structures in their N-termini, favoring a minimal 5’-NNG-3’ PAM preference. Building upon previous successes like iSpyMac, SpRYc emerged from the intelligent exchange of the PID of Sc + + with that of SpRY, creating a chimeric hybrid Cas9.

PAM characterization of SpRYc, utilizing the PAM-SCANR assay and HT-PAMDA, demonstrated its robust binding to PAM sequences, suggesting potential optimization as a “dead” or nickase variant. Despite slower cleavage rates than SpRY, SpRYc showcased broader editing capabilities, paving the way for its application in both nuclease and non-cleavage editing formats.

In silico modeling of SpRYc, conducted via the SWISS-MODEL server, revealed insights into its PAM targeting mechanisms. The study suggested that Sc + +’s optimized loop establishes nonspecific interactions with the PAM, broadening targeting and reducing the need for specific bases at position 2. Importantly, SpRYc’s interactions did not induce self-editing of gRNA plasmids, addressing concerns associated with PAM-flexible CRISPR enzymes.

The discussion emphasizes the significance of overcoming PAM limitations in CRISPR-Cas9 genome editing. SpRYc, with its flexible PAM preferences, exhibits robust editing capabilities, making it suitable for non-nuclease applications like base editing and CRISPR-mediated regulation. The potential integration of high-fidelity mutations into SpRYc further highlights its promise for improved specificity. The study underscores the role of advanced in silico and in vitro PAM engineering methods, emphasizing the synergy of computational algorithms, directed evolution, and structural modeling in protein design. SpRYc marks a significant step toward the future of integrative protein engineering approaches in genome editing.

Zhao, L., Koseki, S.R.T., Silverstein, R.A. et al. PAM-flexible genome editing with an engineered chimeric Cas9. Nat Commun 14, 6175 (2023). https://doi.org/10.1038/s41467-023-41829-y
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