Journal Article

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis

Hou, Zhonggang, Thomson, James A., Sontheimer, Erik J., Chu, Li-Fang, Howden, Sara E., Propson, Nicholas E., Zhang, Yan

2013

Overview

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.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Animals / bacteria / Bacterial Proteins - metabolism / Base Sequence / Biological Sciences / Biomedical research / Cell Line / Cell lines / DNA / Embryonic stem cells / eukaryotic cells / Gene Deletion / Gene Targeting / genes / Genetic engineering / Genetic Engineering - methods / Genome, Human - genetics / Genomes / Gram-negative bacteria / Humans / Mammals / medicine / Meningitis / Molecular Sequence Data / Neisseria meningitidis / Neisseria meningitidis - metabolism / Plasmids / Pluripotent stem cells / Pluripotent Stem Cells - metabolism / proteins / Ribonucleic acid / RNA / RNA - metabolism / RNA Editing - genetics / RNA, Guide - metabolism / Stem cells / Streptococcus pyogenes / Streptococcus thermophilus