Puzzling question in bacterial immune system answered
Researchers have answered a central question about Cas9, an enzyme that plays an essential role in the bacterial immune system and is fast becoming a valuable tool for genetic engineering: How is Cas9 able to precisely discriminate between non-self DNA that must be degraded and self DNA that may be almost identical within genomes that are millions to billions of base pairs long.
A central question has been answered regarding a protein that plays an essential role in the bacterial immune system and is fast becoming a valuable tool for genetic engineering. A team of researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley have determined how the bacterial enzyme known as Cas9, guided by RNA, is able to identify and degrade foreign DNA during viral infections, as well as induce site-specific genetic changes in animal and plant cells. Through a combination of single-molecule imaging and bulk biochemical experiments, the research team has shown that the genome-editing ability of Cas9 is made possible by the presence of short DNA sequences known as “PAM,” for protospacer adjacent motif.”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,” says 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.”With genetically engineered microorganisms, such as bacteria and fungi, playing an increasing role in the green chemistry production of valuable chemical products including therapeutic drugs, advanced biofuels and biodegradable plastics from renewables, Cas9 is emerging as an important genome-editing tool for practitioners of synthetic biology.”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,” says Doudna who holds joint appointments with Berkeley Lab’s Physical Biosciences Division and UC Berkeley’s Department of Molecular and Cell Biology and Department of Chemistry, and is also an investigator with the Howard Hughes Medical Institute (HHMI).Doudna is one of two corresponding authors of a paper describing this research in the journal Nature. The paper is titled “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.” The other corresponding author is Eric Greene of Columbia University. Co-authoring this paper were Samuel Sternberg, Sy Redding and Martin Jinek.Bacterial microbes face a never-ending onslaught from viruses and invasive snippets of nucleic acid known as plasmids. To survive, the 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, (“Cas” stands for CRISPR-associated), 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. There are three distinct types of CRISPR-Cas immunity systems. …
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