Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants
Russell T. Walton, Kathleen A. Christie, Madelynn N. Whittaker, Benjamin P. Kleinstiver
Abstract
Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.
Topics & Concepts
CRISPRCas9NucleaseGenome editingComputational biologyGenome engineeringGenomeBiologyDNALimitingGeneticsGeneMechanical engineeringEngineeringCRISPR and Genetic EngineeringRNA and protein synthesis mechanismsInsect symbiosis and bacterial influences