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 II-A CRISPR-associated enzyme through identification of the natural 5^{prime}-NAAN-3^{prime} PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells.
Highlights
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
CRISPR-associated (Cas) endonucleases are collectively restrained from localizing to any position along double-stranded DNA due to their requirement for targets to neighbor a protospacer adajacent motif (PAM)[2,3,4]
We introduce an ortholog of the well-established Cas[9] from Streptococcus pyogenes (SpyCas9), derived from Streptococcus macacae NCTC 11558, that can instead recognize a short 50-NAA-30 PAM12
Summary
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 introduce an ortholog of the well-established Cas[9] from Streptococcus pyogenes (SpyCas9), derived from Streptococcus macacae NCTC 11558, that can instead recognize a short 50-NAA-30 PAM12 These sequences constitute 18.6% of the human genome, making adjacent adenines the most abundant dinucleotide (Supplementary Fig. 1A–B). The importance of this alternative PAM recognition for a Cas[9] enzyme is reinforced by recent work exposing that Type-V DNA-targeting CRISPR nucleases (including Cas[12] and Cas[14] orthologs), while targeting dsDNA at AT-rich PAM sites with intrinsic high fidelity, will indiscriminately digest single-stranded DNA (ssDNA) once bound to their targets[13,14,15,16]. Our results demonstrate complete 50-NAAN-30 PAM recognition of our engineered variants in all tested contexts
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