Puzzling Question in Bacterial Immune System Answered (http://newscenter.lbl.gov/news-releases/2014/01/29/puzzling-question-in-cas9-answered/)
Berkeley Researchers Uncover the Key to Self-Awareness in Genome Editor
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
"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
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
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
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. Doudna and her research group have focused on the Type II system which relies exclusively upon RNA-programmed 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 recognize matching DNA targets in the context of an entire genome, the classic needle in a haystack problem," says
Doudna, Sternberg and their colleagues used a unique DNA curtains assay and total internal reflection fluorescence microscopy (TIRFM) to image single molecules of Cas9 in real time as they bound to and interrogated DNA. The DNA curtains technology provided unprecedented insights into the mechanism of the Cas9 target search process. Imaging results were verified using traditional bulk biochemical assays.
"We found that Cas9 interrogates DNA for a matching sequence using RNA-DNA base-pairing only after recognition of the PAM, which avoids accidentally targeting matching sites within the bacterium's own genome," Sternberg says. "However, even if Cas9 somehow mistakenly binds to a matching sequence on its own genome, the catalytic nuclease activity is not triggered without a PAM being present. With this mechanism of DNA interrogation, the PAM provides two redundant checkpoints that ensure that Cas9 can't mistakenly destroy its own genomic DNA."
This research was primarily supported by grants from the
For more about the research of
For more about the research of
Most Popular Stories
- Obama Administration Releases Proposal to Regulate For-Profit Colleges
- Apple, HP, Intel May Take a Hit from Slowdown in Smartphone Sales Growth
- Elizabeth Vargas' Husband Marc Cohn Addresses Rumors
- Keurig Adds Peet's coffee, Alters Starbucks deal
- U.S. to Relinquish Gov't Control Over Internet
- Motley Crue's Nikki Sixx Marries Model Courtney Bingham
- FDIC Files Lawsuit on Behalf of Banks Allegedly Hurt by Libor Scandal
- Chinese e-Commerce Giant Alibaba Gears for IPO in U.S.
- Quiznos Files for Chapter 11
- Some California Cities Seeking Water Independence