Efficient genome engineering in human pluripotent stem cells using Cas9 from <i>Neisseria meningitidis</i>

TLDR

Genome engineering in human pluripotent stem cells promises biomedical advances, yet only two CRISPR‑Cas systems (SpCas9 and StCas9) have been adapted for eukaryotic editing, and little is known about HDR‑mediated targeting with long donor templates. The study demonstrates efficient HDR‑based targeting of an endogenous gene in three hPSC lines using a Neisseria meningitidis CRISPR‑Cas system. NmCas9 uses a distinct 5′‑NNNNGATT‑3′ PAM, enabling RNA‑guided editing that differs from SpCas9 and StCas9. NmCas9 functions with a single‑guide RNA and its unique PAM expands editable sequence contexts compared to SpCas9.

Abstract

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.