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

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 activ

SUBMITTER: Ageely EA 

PROVIDER: S-EPMC8593028 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature