Scientists have tweaked a key region within the Cas9 protein to produce a highly accurate gene editor with the lower levels of off-target cutting than current methods. The scientists from the University of California, Berkeley and Massachusetts General Hospital reported their results yesterday in advance of publication in the journal Nature.
The protein domain identified is called REC3 and it governs how accurately CRISPR-Cas9 homes in on a target DNA sequence. The researchers were able to introduce mutations into REC3 and test for ones that increased accuracy without interrupting function.
The discovery was preempted by the recent creation of highly accurate Cas9 proteins, eSpCas9(1.1) and SpCas9-HF1. Researchers in Jennifer Doudna’s lab at UC Berkeley sought to understand why they cut with higher specificity than wild-type Cas9.
In the current study, the researchers used single-molecule FRET to measure how various domains in the Cas9-sgRNA protein complex—particularly 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.
Image: The Cas9 protein (gray) is an RNA-guided nuclease that can be programmed to bind and cut any matching DNA sequence (dark blue double helix), making it a powerful tool for genome engineering. Upon target binding, Cas9 protein domains undergo conformational rearrangements (the motions of individual amino acids are represented by rocket tails) to activate the Cas9-sgRNA complex for target cleavage. The REC3 domain (teal) is responsible for target sensing, which signals the outward rotation of the REC2 domain (magenta) to open a path for the HNH nuclease domain (yellow). This active conformation of Cas9 is then capable of triggering concerted cleavage of both strands of the target DNA. Image courtesy of Janet Iwasa graphic for Doudna Lab.