Structure-guided mutagenesis has guided the engineering of an “enhanced specificity” Streptococcus pyogenes Cas9. In the newly engineered Cas9, three positively charged amino acids from the enzyme’s positively charged, DNA-cradling groove have been replaced by neutral amino acids. With the modified Cas9, the binding of on-target sites appears to be weakened much less than the binding of off-target sites. [Ian Slaymaker, Broad Institute]
The CRISPR-Cas9 gene-editing system has been known to be overzealous, unwilling to call it quits after cutting its target site. Unfortunately, in many cases, CRISPR-Cas9 won’t leave well enough alone. It persists inside a cell, cutting additional sites, introducing unwanted edits, and propagating “off target” effects.
A calmer CRISPR-Cas9, reasoned scientists at the Broad Institute, might make a finer editing tool, one less given to coloring outside the lines. These scientists, led by Feng Zhang, Ph.D., started tinkering with the CRISPR-Cas9’s business end, the DNA-snipping Cas9 endonuclease. Ultimately, they developed a Cas9 that binds more weakly, whether its binding partner is an on-target or off-target stretch of DNA. Yet they were able to ensure that the binding of on-target sites was weakened much less than the binding of off-target sites.
The approach used by Dr. Zhang and colleagues involved swapping out some of Cas9’s 1,400-or-so amino acids. Cas9 has positively charged amino acids that form a positively charged groove that cradles positively charged stretches of DNA. By removing some of these positively charged amino acids and replacing them with neutral amino acids, the scientists hoped to create a less grabby, cut-happy Cas9.
After experimenting with various possible changes, Dr. Zhang's team found that mutations in three amino acids dramatically reduced off-target cuts. For the guide RNAs tested, off-target cutting was so low as to be undetectable.
Details about this work appeared December 1 in the journal Science, in an article entitled, “Rationally engineered Cas9 nucleases with improved specificity.” The article describes how Dr. Zhang’s team used structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9).
“Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that 'enhanced specificity' SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage,” wrote the article’s authors. “Thus, eSpCas9 could be broadly useful for genome editing applications requiring a high level of specificity.”
The Zhang lab is immediately making the eSpCas9 enzyme available for researchers worldwide. The team believes the same charge-changing approach will work with other recently described RNA-guided DNA targeting enzymes, including Cpf1 as well as C2c1, C2c2, and C2c3, which Dr. Zhang and his collaborators reported on earlier this year.
The prospect of rapid and efficient genome editing raises many ethical and societal concerns, said Dr. Zhang. “Many of the safety concerns are related to off-target effects,” he continued. “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.”