Puzzling question in bacterial immune system answered
Puzzling question in
bacterial immune system answered
Posted by: Deepak Kumar
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.
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 Samuel Sternberg, lead author of the Nature paper and a
member of Doudna's research group. "All of the scientists who are
developing RNA-programmable Cas9 for genome engineering are relying on its
ability to target unique 20-base-pair long sequences inside the cell. However,
if Cas9 were to just blindly bind DNA at random sites across a genome until
colliding with its target, the process would be incredibly time-consuming and
probably too inefficient to be effective for bacterial immunity, or as a tool
for genome engineers. 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 non-target DNA sites."
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."
Note: An educational video to describing the research is
available at: http://www.youtube.com/watch?v=M739wgbcKuA
Story Source:
The above story is based on materials provided by
DOE/Lawrence Berkeley National Laboratory. The original article was written by
Lynn Yarris. Note: Materials may be edited for content and length.
Journal Reference:
Samuel H. Sternberg, Sy Redding, Martin Jinek, Eric C.
Greene, Jennifer A. Doudna. DNA interrogation by the CRISPR RNA-guided
endonuclease Cas9. Nature, 2014; DOI: 10.1038/nature13011