New Genome Engineering Technique Eases Gene Repair in Human Pluripotent Stem Cells

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.

The CRISPR/Cas (CRISPR associated) system is a new genome-engineering technology recently developed from prokaryotes' adaptive immune response systems. The CRISPR system uses a short, noncoding RNA (crRNA) to target a human codon-optimized Cas9 nuclease to complementary (protospacer) sequences in the host genome. These sequences or arrays are composed of direct repeats that are separated by similarly sized nonrepetitive spacers. CRISPR arrays, together with a group of associated proteins, confer resistance to phages, possibly by an RNA-interference-like mechanism.

Investigators at the University of Wisconsin (Madison USA) and their colleagues at Northwestern University (Evanston, IL, USA) used a CRISPR-Cas system identified in the bacterium Neisseria meningitidis, which is distinct from the commonly used Streptococcus pyogenes system, to demonstrate efficient genome engineering in human pluripotent stem cells (hPSCs).

Heretofore, only two CRISPR-Cas systems (from S. pyogenes and S. thermophilus), each with their own distinct targeting requirements and limitations, had been developed for genome editing. In addition, only limited information existed regarding homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems.

The investigators wrote in the August 12, 2013, online edition of the journal Proceedings of the National Academy of Sciences of the United States of America (PNAS) that by employing a distinct CRISPR-Cas system from N. meningitidis, they were able to demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognized a 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 was 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 increased the sequence contexts amenable to RNA-directed genome editing.

“Human pluripotent stem cells can proliferate indefinitely and they give rise to virtually all human cell types, making them invaluable for regenerative medicine, drug screening, and biomedical research,” said senior author Dr. James A. Thomson, professor of embryonic stem cell biology at the University of Wisconsin. “Our collaboration with the Northwestern team has taken us further toward realizing the full potential of these cells because we can now manipulate their genomes in a precise, efficient manner. With this system, there is the potential to repair any genetic defect, including those responsible for some forms of breast cancer, Parkinson’s, and other diseases.”

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University of Wisconsin
Northwestern University