Scientists from the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley say they have worked out how the bacterial enzyme Cas9, guided by RNA, is able to identify and degrade foreign DNA during viral infections. They add they now also have a better understanding of how Cas9 can induce site-specific genetic changes in animal and plant cells.
“Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites,” write the investigators in this week’s issue of Nature. “We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Nontarget DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA.”
“Our results reveal two major functions of the PAM that explain why it is so critical to the ability of Cas9 to target and cleave DNA sequences matching the guide RNA,” explained Jennifer Doudna, Ph.D., a UC biochemist who led the study, which is entitled “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.” “The presence of the PAM adjacent to target sites in foreign DNA and its absence from those targets in the host genome enables Cas9 to precisely discriminate between non-self DNA that must be degraded and self DNA that may be almost identical. The presence of the PAM is also required to activate the Cas9 enzyme.”
Cas9 is emerging as an important genome-editing tool for practitioners of synthetic biology, according to Dr. Doudna.
“Understanding how Cas9 is able to locate specific 20-base-pair target sequences within genomes that are millions to billions of base pairs long may enable improvements to gene targeting and genome editing efforts in bacteria and other types of cells,” she said.
To defend against foreign DNA, microbes deploy an adaptive nucleic acid-based immune system that revolves around a genetic element known as CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats. Through the combination of CRISPRs and RNA-guided endonucleases such as Cas9, bacteria are able to utilize small customized crRNA molecules (for CRISPR RNA) to guide the targeting and degradation of matching DNA sequences in invading viruses and plasmids to prevent them from replicating.
“What has been a major puzzle in the CRISPR-Cas field is how Cas9 and similar RNA-guided complexes locate and recognize matching DNA targets in the context of an entire genome, the classic needle in a haystack problem,” pointed out Samuel Sternberg, Ph.D., lead author of the Nature paper and a grad student member of Dr. Doudna's research group. “Our study shows that Cas9 confines its search by first looking for PAM sequences. This accelerates the rate at which the target can be located, and minimizes the time spent interrogating nontarget DNA sites.”