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A CRISPR system built to use the FnCas9 enzyme was found to edit genomes more efficiently and with less unintended damage than existing technologies, researchers at CSIR-Institute of Genomics and Integrative Biology, New Delhi
CRISPR-Cas9
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a gene editing technology, which replicates natural defence mechanism in bacteria to fight virus attacks, using a special protein called Cas9.
It usually involves the introduction of a new gene, or suppression of an existing gene, through a process described as genetic engineering.
CRISPR technology does not involve the introduction of any new gene from the outside.
CRISPR-Cas9 technology is often described as ‘Genetic Scissors’.
Its mechanism is often compared to the ‘cut-copy-paste’, or ‘find-replace’ functionalities in common computer programmes.
A bad stretch in the DNA sequence, which is the cause of disease or disorder, is located, cut, and removed and then replaced with a ‘correct’ sequence.
The tools used to achieve this are biochemical i.e., specific protein and RNA molecules.
The technology replicates a natural defence mechanism in some bacteria that uses a similar method to protect itself from virus attacks.
Mechanism:
The first task is to identify the particular sequence of genes that is the cause of the trouble.
Once that is done, an RNA molecule is programmed to locate this sequence on the DNA strand
After this Cas9 is used to break the DNA strand at specific points, and remove the bad sequence.
A DNA strand, when broken, has a natural tendency to re-attach and heal itself. But if the auto-repair mechanism is allowed to continue, the bad sequence can regrow.
So, scientists intervene during the auto-repair process by supplying the correct sequence of genetic codes, which attaches to the broken DNA strand.
The entire process is programmable, and has remarkable efficiency, though the chances of error are not entirely ruled out.
Limitations
CRISPR-Cas9 system can also recognise and cut parts of the genome other than the intended portion.
Such “off-target” effects are more common when using the SpCas9 enzyme derived from Streptococcus pyogenes bacteria.
Scientists have been able to engineer versions of SpCas9 with higher fidelity but only at the cost of editing efficiency
FnCas9 enzyme
To overcome these issues, researchers are exploring Cas9 enzymes from Francisella novicida bacteria.
While this Cas9, called FnCas9, is highly precise, it has low efficiency as well.
Researchers tinkered with amino acids in FnCas9 that recognise and interact with the protospacer adjacent motif (PAM) sequence on the host genome.
By doing this, increase the binding affinity of the Cas protein with the PAM sequence
The Cas9 can then sit on the DNA in a stronger configuration, and your gene editing becomes much more effective.
Potential applications
Enhanced FnCas9 is a viable option for treating genetic disorders
Enzyme’s efficient at correcting a genetic mutation that causes Leber congenital amaurosis type 2 (LCA2), a form of inherited blindness.
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