Neisseria meningitidis Cas9 (NmeCas9)

Characteristics

Cleaves both strands of the target with its RuvC and HNH nuclease domains, producing a blunt double-strand break; the D16A, D587A, H588A and N611A substitutions together abolish cleavage. The 1,082-amino-acid protein is 286 residues smaller than SpCas9, and its coding sequence of 3.25 kb is 850 bp shorter. Guides carrying 20 to 24 nucleotides of target complementarity edit with comparable efficiency, while 18- and 19-nucleotide guides are markedly less active. Besides the NNNNGATT consensus, the variant PAMs NNNNGCTT, NNNNGTTT, NNNNGACT, NNNNGATA, NNNNGTCT and NNNNGACA also support editing. GUIDE-seq finds no off-target editing above background for 17 of 19 guide RNAs in human cells, and none or one off-target site at two loci where matched SpCas9 guides give 271 and 54.

Applications: Knockout editing in HEK293T and K562 cells, human fibroblasts and mouse embryonic stem cells. HDR knock-in of EGFP at the OCT4 locus reaches about 60% efficiency in the H1 and H9 human embryonic stem cell lines and in an iPS cell line. A single AAV8 vector carrying NmeCas9 and its sgRNA edits Pcsk9 and Hpd in mouse liver after tail-vein injection, lowering serum cholesterol and maintaining body weight in Fah-deficient mice. Catalytically dead NmeCas9 fused to VP64 activates transcription.

Limitations: The NNNNGATT PAM occurs once every 128 bp of genomic DNA against once every 8 bp for the NGG PAM of SpCas9, so many sites are untargetable; Nme2Cas9, a related ortholog of similar size, recognizes an NNNNCC PAM and matches SpCas9 target density. On-target editing in HEK293T cells is generally lower than that of SpCas9. NmeCas9 guide RNAs are orthogonal to those of SpCas9 and cannot be exchanged.

References

1. Esvelt et al. (2013). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods - Esvelt 2013 NmeCas9
2. Lee et al. (2016). The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells. Mol Ther - Lee 2016 NmeCas9 Specificity
3. Amrani et al. (2018). NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome Biol - Amrani 2018 NmeCas9 Fidelity
4. Ibraheim et al. (2018). All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo. Genome Biol - Ibraheim 2018 NmeCas9 AAV
5. Edraki et al. (2019). A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Mol Cell - Edraki 2019 Nme2Cas9