Introduction:
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications…
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How does it work?
The CRISPR-Cas9 system consists of two key molecules that introduce a change (mutation) into the DNA. These are:
- an enzyme called Cas9. This acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed.
- a piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.
How was it developed?
Some bacteria have a similar, built-in, gene editing system to the CRISPR-Cas9 system that they use to respond to invading pathogens like viruses, much like an immune system.
Using CRISPR the bacteria snip out parts of the virus DNA and keep a bit of it behind to help them recognise and defend against the virus next time it attacks.
Scientists adapted this system so that it could be used in other cells from animals, including mice and humans.
What are the applications and implications?
- CRISPR-Cas9 has a lot of potential as a tool for treating a range of medical conditions that have a genetic component, including cancer, hepatitis B or even high cholesterol.
- Many of the proposed applications involve editing the genomes of somatic (non-reproductive) cells but there has been a lot of interest in and debate about the potential to edit germline (reproductive) cells.
- Because any changes made in germline cells will be passed on from generation to generation it has important ethical implications.
- Carrying out gene editing in germline cells is currently illegal in the UK and most other countries.
- By contrast, the use of CRISPR-Cas9 and other gene editing technologies in somatic cells is uncontroversial. Indeed they have already been used to treat human disease on a small number of exceptional and/or life-threatening cases.
T7 Endonuclease :
It recognizes and cuts mismatched DNA, cruciform DNA, Holliday structures or junctions, and heteroduplex DNA. It can also recognize and cut double-stranded DNA with nicks with a lower speed. The enzyme cuts the first, second or third phosphodiester bond that is 5′ to the mismatch.
Recombinant T7 Endonuclease I is purified from E.coli expression system. It has high purity and can be applicable for gene mutation, SNP and TALEN or CRISPR/Cas9-mediated mutation detection. It can recognize DNA mismatches, resolve four-way junction or branched DNA, detect heteroduplex or nicked DNA and randomly cleave linear DNA for shot-gun cloning.