Technical roadblock to application of CRISPR-Cas9 genome editing addressed

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT (MA, USA) engineer changes to the CRISPR-Cas9 genome editing system that decrease ‘off-target’ editing errors.

Go to the profile of Alexandra Thompson
Dec 08, 2015

The CRISPR-Cas9 system makes precisely targeted modifications in a cell’s DNA by using a short RNA sequence matching the target site to bind and allow the enzyme Cas9 to alter the DNA. However, once inside a cell, Cas9 can bind to and cut additional off-target sites and could therefore result in unwanted edits that can alter gene expression or knock a gene out entirely, potentially resulting in cancer or other issues.

In a newly published paper, Feng Zhang and colleagues reported that off-target editing can be reduced to almost undetectable levels by changing three of the approximately 1400 amino acids that make up the Cas9 enzyme, which comes from S. pyogenes. They used knowledge about the structure of the Cas9 protein to hypothesize that replacing some of the positively charged amino acids with neutral ones would decrease the binding of off-target sequences much more than on-target sequences.

After testing different alterations the scientists found that mutations in three amino acids resulted in a dramatic decrease in off-target edits to levels undetectable by the guide RNAs tested. Therefore, the newly engineered enzyme, named eSpCas9, will be useful for genome editing applications that require a high level of specificity. However, according to Zhang, who spoke at the International Summit on Gene Editing in Washington, DC, USA, the ability to quickly and efficiently edit genomes raises many ethical and societal concerns. “Many of the safety concerns are related to off-target effects,” he explained. “We hope the development of eSpCas9 will help address some of those concerns, but we certainly don’t see this as a magic bullet. The field is advancing at a rapid pace, and there is still a lot to learn before we can consider applying this technology for clinical use.”

The Zhang lab has already made the enzyme available for researchers worldwide to use, and believes the same charge-changing approach will work with other recently described RNA-guided DNA targeting enzymes, including Cpf1, C2C1, and C2C3, which Zhang and his collaborators reported on earlier this year.

Source:; Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science doi:10.1126/science.aad5227 (2015) (Epub ahead of print).

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Alexandra Thompson

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