CRISPR-Cas Systems Reduced to a Minimum

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 (N(4)CC) 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.