Cas9’s secrets revealed
26 Feb 2014 by Evoluted New Media
Bacterial enzyme Cas9 needs short DNA sequences to identify and degrade foreign DNA during viral infections American researchers have discovered.
Cas9 (CRISPR-associated) is guided by RNA, but needs short DNA sequences known as PAM (protospacer adjacent motif) in order to function say the team from the Berkeley Laboratory and University of California Berkeley. They used single-molecule imaging and bulk biochemical experiments to reveal their result, published in Nature.
“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,” said Jennifer Doudna, the biochemist who led this study. “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 synthetic biology so understanding how it locates 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, said Doudna.
In order to survive the never-ending onslaught from viruses and plasmids, bacterial microbes use an adaptive nucleic acid-based immune revolving around CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats. Combined with RNA-guided endonucleases like Cas9, bacteria use small customised crRNA (CRISPR RNA) to guide targeting and degradation of matching DNA sequences in invading enemies to prevent replication. There are three distinct types of CRISPR–Cas immunity systems; Doudna’s group focused on the Type II system which relies on Cas9 to cleave double-stranded DNA at target sites.
“What has been a major puzzle in the CRISPR–Cas field is how Cas9 and similar RNA-guided complexes locate and recognise matching DNA targets in the context of an entire genome,” says Samuel Sternberg.
Sternberg says that scientists developing RNA-programmable Cas9 for genome engineering rely on its ability to target unique sequences inside the cell. The study shows that Cas9 confines its search by first looking for PAM sequences, which accelerates the rate at which the target can be located, and minimises the time spent interrogating non-target DNA sites.
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
