CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However， in?vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors)， off-target editing， or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here， we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (NCC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient geno... More
CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However， in?vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors)， off-target editing， or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here， we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (NCC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility， exceptional editing accuracy within cells， and high target site density for in?vivo genome editing applications.
