CRISPR / Cas9 technology explained, CRISPR is a simple yet powerful tool for genome editing. Researchers can modify the gene function by easily altering DNA sequences. Its many applications include genetic defect correction, disease treatment and prevention and crop improvement. Its promise, however, also raises ethical issues.
“CRISPR” (pronounced “crisper”) is commonly referred to as the abbreviation for “CRISPR-Cas9.” The protein Cas9 is an enzyme which acts like a pair of molecular scissors that cut DNA strands. This enzyme is a “CRISPR” protein.
CRISPR technology is adapted from the bacteria and archaea (so local domain of microorganisms) natural defence mechanisms. The RNA from CRISPR and different Cas proteins such as Cas9 is used by these organisms for foil attacks by viruses and other foreign bodies. They mainly do so by cutting down a foreign invader’s DNA and destroying that. When transferring these components to other, more complex organisms, they enable gene manipulation or “editing.“
Nobody knew how the process looked until 2017. A team of researchers led by Mikihiro Shibata from Kanazawa University and Hiroshi Nishimasu from Tokyo’s University have demonstrated how the first time a CRISPR takes action in a paper published 10 November 2017 in the journal Nature Communications.
What exactly is CRISPR-Cas9?
CRISPRs: “CRISPR” stands for “clusters of regularly interspaced short palindromic repeats.”
It is a specialized DNA area with 2 distinct features: the presence of repeats and spacers of nucleotides. Repeated nucleotide sequences — DNA building blocks — are dispersed in a CRISPR region. Spacers are DNA bits which are split between these repeated sequences.
In the case of bacteria, the spacers are derived from viruses that attacked the body previously. They serve as a memory bank that enables bacteria to identify viruses and combat future attacks.
Rodolphe Barrangou and a team of researchers from Danisco, a food ingredients company, demonstrated this for the first time experimentally. The researchers used Streptococcus thermophilus bacteria, often found in yoghurt and other dairy cultivations, to be their model in a paper published in Science in 2007.
They observed that new spacers were added to the CRISPR area following a virus attack. In addition, these spacers had a DNA sequence identical to those of the virus genome. They also manipulated the distances by removing or placing new sequences of viral DNA.
This enabled them to modify the resistance of the bacteria to a certain virus attack. Thus, the researchers confirmed that CRISPRs play a role in regulating bacterial immunity.
The CRISPR RNA is transcribable and processed in a CRISPR RNA, or crRNA, once spacers are integrated and the virus attacks are again, and the CRISPR sequence functions as the template for creating a complementary single-stranded RNA sequence. In a 2014 review, Jennifer Doudna and Emmanuelle Charpentier published in the journal Science, each CRNA consists of a nucleotide repeat and a spacer portion.
Cas9: The Cas9 protein is an enzyme that cuts foreign DNA.
This protein typically binds to two RNA molecules: crRNA and one called tracrRNA. The two of them then lead Cas9 to the target site. This DNA expansion complements a 20-nucleotide crRNA stretch.
Cas9 cuts both strands of the DNA double helix using two separated regions or “domains” on its structure to produce what is called “double-stranded break,” the article from the Science article of 2014 states.
There is an integrated safety mechanism that ensures that Cas9 is not just cut in a genome. Short DNA sequences known as PAMs serve as tags and are next-door to the DNA sequence of the target. If Cas9 does not see a PAM next door to its target DNA sequence, it will not be shortened. According to a review published in Nature Biotechnology, Cas9 never attacks the CRISPR region in bacteria.
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