Most Accurate CRISPR Yet- Key Region The Editing System Discovered
The RNA-guided CRISPR–Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. And in order to minimize the chance that CRISPR-Cas9 will edit DNA at the wrong place, scientists have now designed a version of Cas9, the enzyme that cuts DNA that avoids mistakes with unprecedented precision.
Currently, scientists using CRISPR-Cas9 create a single-guide ribonucleic acid, or sgRNA, an RNA molecule that includes a chain of 20 ribonucleic acids that complements a specific 20-nucleic-acid DNA sequence they want to target, and attach it to Cas9. This guide RNA allows Cas9 to home in on the complementary DNA, bind to it and cut the double stranded helix. However, the Cas9-sgRNA complex can bind to DNA that doesn’t exactly match, leading to undesirable off-target cutting.
The researchers at the University of California, Berkeley, and Massachusetts General Hospital said they have tweaked the region within the Cas9 protein to produce a hyper-accurate gene editor with the lowest level of off-target cutting to date.
The protein domain the researchers identified as a master controller of DNA cutting is an obvious target for re-engineering to improve accuracy even further, the researchers say
. Mutating one piece of the nuclease, called REC3, reduced off-target effects to below that of other high-fidelity Cas9 enzymes.“If you mutate certain amino acid residues in REC3, you can tweak the balance between Cas9 on-target activity and improved specificity,” coauthor Janice Chen, a graduate student in Jennifer Doudna’s lab at the University of California, Berkeley, says in a press release. “We were able to find the sweet spot where there is sufficient activity at the intended target but also a large reduction in off-target events,”
In the current study supported by the U.S. National Institutes of Health (NIH) and the U.S. National Science Foundation (NSF), Chen and her colleagues used a technique called single-molecule Forster resonance energy transfer (FRET) to precisely measure how the various protein domains in the Cas9-sgRNA protein complex – in particular REC3, REC2 and HNH – move when the complex binds to DNA.
They first determined that the increased eSpCas9(1.1) and SpCas9-HF1 specificity could be explained by their higher threshold for the HNH conformational switch, making them less likely to activate scissors when bound to an off-target sequence. They then found REC3 to be responsible for setting the accuracy of target binding, signaling the outward rotation of the REC2 domain to open a path for the HNH nuclease domain.
Using their newfound knowledge, they engineered a hyper-accurate Cas9 dubbed Hypa Cas9 that retains on-target efficiency but is slightly better at discriminating between on and off target sites. The researchers believe this process of mutating REC3 could be repeated in the future to create even more accurate confirmations.
“If you mutate certain amino acid residues in REC3, you can tweak the balance between Cas9 on-target activity and improve specificity; we were able to find the sweet spot where there is sufficient activity at the intended target but also a large reduction in off-target events,” Chen was quoted as saying in the news release from UC Berkeley.
“We have found that even minor alterations in the REC3 domain of Cas9 affect the differential between on- and off-target editing, which suggests that this domain is an obvious candidate for in-depth mutagenesis to improve targeting specificity,” Chen says in the release. “As an extension of this work, one could perform a more unbiased mutagenesis within REC3 than the targeted mutations we have made.”
