Genome engineering in human pluripotent stem cells (hPSCs) holds great promise 
for biomedical research and regenerative medicine. Recently, an RNA-guided, 
DNA-cleaving interference pathway from bacteria [the type II clustered, 
regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated 
(Cas) pathway] has been adapted for use in eukaryotic cells, greatly 
facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus 
pyogenes and Streptococcus thermophilus), each with their own distinct targeting 
requirements and limitations, have been developed for genome editing thus far. 
Furthermore, limited information exists about homology-directed repair 
(HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these 
systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis, 
we demonstrate efficient targeting of an endogenous gene in three hPSC lines 
using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) 
recognizes a 5'-NNNNGATT-3' protospacer adjacent motif (PAM) different from 
those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 
and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a 
single-guide RNA (sgRNA) to direct its activity. Because of its distinct 
protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases 
the sequence contexts amenable to RNA-directed genome editing.