How does the Cas9 protein "recognize" the desired section of DNA
The main mystery of the bacterial immune system has been solved
NanoNewsNet based on Berkeley Lab materials: Puzzling Question in Bacterial Immune System AnsweredScientists have received an answer to the main question about a protein that plays a significant role in the immune system of bacteria and is rapidly becoming a valuable tool of genetic engineering.
A group of researchers from Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California at Berkeley (University of California, Berkeley) has established how the bacterial enzyme Cas9, guided by RNA, identifies and destroys foreign DNA in viral infections, and also causes site-specific genetic changes in animals and plants cages. As scientists have found out, the ability of Cas9 to edit the genome is realized due to the presence of short DNA sequences known as PAM (protospacer adjacent motif).
"Our study reveals two main functions of the PAM motif, which explain why it is so important for the realization of Cas9's ability to target and cleave DNA sequences complementary to its guiding RNA," says biochemist Jennifer Doudna, PhD, head of the study. "The presence of a FRAME adjacent to target sites in foreign DNA and its absence from these targets in the genome of the host organism allows Cas9 to accurately distinguish between "not its" DNA, which should be destroyed, and "its" DNA, which may be almost identical. In addition, the presence of PAM is necessary for the activation of the Cas9 enzyme."
A short DNA sequence (trinucleotide) known as PAM (shown in yellow),
allow the bacterial Cas9 enzyme to detect and destroy foreign DNA, as well as
cause site-specific genetic changes in animal and plant cells.
The presence of PAM is also required for the activation of Cas9. (Fig. KC Roeyer)
With the creation of genetically engineered microorganisms, such as bacteria and fungi, playing an increasing role in the production of valuable products by green chemistry methods, including therapeutic drugs, biofuels and biodegradable plastics, the Cas9 enzyme is becoming an important genome editing tool in the hands of practitioners in the field of synthetic biology.
"Understanding how Cas9 finds specific target sequences consisting of 20 base pairs in a genome consisting of millions and billions of base pairs will improve gene targeting and genome editing of bacteria and other cell types," Dr. Dudna continues. Dr. Dudna combines work in the Department of Physical Biosciences (Physical Biosciences Division) of the Berkeley Laboratory with teaching at the Departments of Molecular and Cellular Biology and Chemistry of the University of California at Berkeley, and is also a researcher at the Howard Hughes Medical Institute.
Bacteria resist the never-ending onslaught from viruses and invasive nucleic acid fragments known as plasmids. To survive, microbes use an adaptive nucleic acid-based immune system revolving around a genetic element known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Thanks to the combination of CRISPRs and RNA-guided endonucleases, such as Cas9 (Cas – CRISPR-associated), bacteria can use small specialized crRNA (or CRISPR RNA) molecules to target and cleave complementary DNA sequences when viruses and plasmids invade and prevent their replication. There are three different types of CRISPR-Cas immune systems. Dr. Dudna and her group focused their attention on the type II system, which is based exclusively on RNA-programmed Cas9 in cleavage by target sites of double-stranded DNA.
"What has always been one of the main puzzles in the field of CRISPR-Cas is how Cas9 and similar RNA-guided complexes detect and recognize complementary DNA targets in the context of the entire genome - a classic needle–in–a-haystack problem," says Samuel Sternberg, PhD, lead author of the paper about the study published in the journal Nature (DNA interview by the CRISPR RNA-guided endonuclease Cas9). "All scientists developing RNA-programmable Cas9 for genetic engineering rely on its ability to target unique intracellular sequences with a length of 20 base pairs. However, if Cas9 were to just blindly bind DNA at random locations in the genome before colliding with its target, the process would be incredibly time-consuming and probably too inefficient to provide immune protection to the bacterium or be used as a tool in genetic engineering. Our research shows that Cas9 restricts its search by finding PAM sequences first. This increases the speed of finding the target and minimizes the time spent on rechecking non-target DNA sites."
To visualize individual Cas9 molecules in real time during their search and "study" of DNA, scientists used a unique method of DNA analysis – the so-called DNA curtains technology – and fluorescence microscopy of total internal reflection (total internal reflection fluorescence microscopy, TIRFM).
The DNA curtain method allows us to achieve an unprecedented understanding of the mechanism of the Cas9 enzyme's search for its target.
"We found that Cas9 rechecks DNA to find the appropriate sequence by forming RNA-DNA base pairs only after recognizing the frames, which avoids accidental targeting of complementary sites of the bacterium's own genome," Dr. Sternberg continues. "However, even if Cas9 somehow mistakenly binds to the complementary sequence of the bacterium's own genome, its catalytic nuclease activity will not turn on without the presence of PAM. With this DNA evaluation mechanism, PAM provides two backup checkpoints to ensure that Cas9 cannot mistakenly destroy the DNA of the bacterium's own genome."
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