Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

• Published in: Science (American Association for the Advancement of Science) Vol. 368; no. 6488; pp. 290 - 296
• Main Authors: Walton, Russell T, Christie, Kathleen A, Whittaker, Madelynn N, Kleinstiver, Benjamin P
• Format: Journal Article
• Language: English
• Published: United States The American Association for the Advancement of Science 17-04-2020
• Subjects: CRISPR; CRISPR-Associated Protein 9 - chemistry; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; Editing; Enzymes; Gene Editing - methods; Gene sequencing; Gene Targeting - methods; Genetic Predisposition to Disease; Genetic variance; Genome editing; Genomes; HEK293 Cells; Humans; Literary Devices; Mutagenesis; Mutation; Nuclease; Protein Domains; Streptococcus pyogenes; Substrate Specificity; Target recognition
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.aba8853

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of
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.