UC Berkeley

The ongoing battle between bacteria and phage has influenced the evolution of complex defense pathways. In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated (Cas) proteins form the basis for RNA-guided adaptive immunity and antiviral defense. The most streamlined CRISPR systems contain the class 2 interference cas genes, which encode a single polypeptide capable of executing all the functions of target search and destruction. This work describes the mechanisms of target recognition and cutting by the class 2 DNA-targeting nucleases, Cas9 and Cas12a (formerly Cpf1), with an emphasis on their biotechnological applications.

The ability to program Cas9 or Cas12a to target and cut any DNA sequence by simply changing its guide RNA sequence has revolutionized genome editing. Although Cas9 and Cas12a are both RNA-guided DNA endonucleases, these proteins have little sequence or structural similarity, and several functional differences highlight the evolutionary diversification of CRISPR interference proteins. In particular, Cas9 contains two nuclease domains called HNH and RuvC that cut each strand of the dsDNA substrate to generate a blunt end. The finding that Cas9 undergoes multiple conformational states in the steps leading up to DNA cleavage underscored the importance of conformational control. Using biochemical and single-molecule approaches, we describe a conformational checkpoint that governs activation of Cas9 and uncover a region within the protein responsible for sensing target mismatches that can be further engineered to enhance cleavage fidelity.

Unlike Cas9, Cas12a cuts dsDNA with a single RuvC nuclease domain, leaving a staggered end. We find that the RuvC is active only on single-stranded DNA (ssDNA), highlighting the requirement for dsDNA unwinding to facilitate cutting of both strands of the dsDNA substrate. Furthermore, we reveal that once Cas12a binds to a complementary ss- or dsDNA sequence, the RuvC nuclease non-specifically degrades any ssDNA molecule in trans. In the presence of a reporter molecule, this trans-cleavage activity by Cas12a is repurposed as a versatile DNA detection tool and has attomolar sensitivity when coupled with isothermal amplification. Together, the mechanistic framework of Cas9 and Cas12a provides a foundation for engineering fidelity and developing novel DNA detection technologies.