A Cas9 with Complete PAM Recognition for Adenine Dinucleotides

bioRxiv preprint first posted online Sep. 27, 2018; doi: http://dx.doi.org/10.1101/429654. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
Authors
Noah Jakimo , Pranam Chatterjee , Lisa Nip , & Joseph M Jacobson
10-24-2018 HSW 1057
12:00pm
PST
Categories
RNA Modification & Editing
Speaker
Stacey Phan
Abstract

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a Type-IIA CRISPR-associated enzyme through identification of the natural 5’-NAA-3’ PAM specificity of a Streptococcus macacae Cas9 (Smac Cas9). We further recombine protein domains between Smac Cas9 and its well-established ortholog from Streptococcus pyogenes (Spy Cas9), as well as an “increased" nucleolytic variant (iSpy Cas9), to achieve consistent mediation of gene modification and base editing. In a comparison to previously reported Cas9 and Cas12a enzymes, we show that our hybrids recognize all adenine dinucleotide PAM sequences and possess robust editing efficiency in human cells.