Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

Eman A. Ageely, Ramadevi Chilamkurthy, Sunit Jana, Leonora Abdullahu, Daniel O’Reilly, Philip J. Jensik, Masad J. Damha () and Keith T. Gagnon ()
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Eman A. Ageely: Southern Illinois University
Ramadevi Chilamkurthy: School of Medicine, Southern Illinois University
Sunit Jana: McGill University
Leonora Abdullahu: McGill University
Daniel O’Reilly: McGill University
Philip J. Jensik: School of Medicine, Southern Illinois University
Masad J. Damha: McGill University
Keith T. Gagnon: Southern Illinois University

Nature Communications, 2021, vol. 12, issue 1, 1-15

Abstract: Abstract CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

Date: 2021
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DOI: 10.1038/s41467-021-26989-z

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