CRISPR is the abbreviation of clustered regularly interspaced short palindromic repeats, a mediated defense mechanism adopted by prokaryotes.The widely applied CRISPR technology is the Crispr/Cas system for gene sequence editions by transferring Cas9 protein family encoded gene into cells and potentially simplifies the procedure of cutting DNA at a desired location . Cas9 was first used for targeted genome editing in prokaryotes by Prof. Emmanuelle Charpentier & Prof. Jennifer Doudna (2012) and later adapted for use in eukaroytes by Prof. Feng Zhang (2013).
There are three CRISPR systems, one of which has been adapted for eukaryotes. This is the Type II CRISPR system, which involves Cas9 protein, and it consists of three stages summarised briefly below:
- Acquisition, where the invading DNA is recognized by Cas 1 and 2, and a protospacer is cleaved. The protospace then ligates to the repeat directly opposite the leader sequence, and single-stranded extension repairs the CRISPR array and duplicates the repeat (forming pre-crRNA). A copy of the invader's DNA is now incorporated into CRISPR's locus.
- crRNA processing, where tracrRNA hybridizes to the repeat regions of the pre-crRNA, and with the help of RNAse III, mature crRNAs with individual spacer sequences are created. Note: there will be a secondary trimming performed at the 5'-end to produce mature crRNAs.
- Interference, where the mature crRNA:tracrRNA complex directs cas9 to the target DNA via base pairing between the spacer on the crRNA and the protospacer on the target DNA next to the PAM (protospacer adjacent motif). Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.
The DNA repairs this double stranded break (DSB) by either non-homologous end joining (NHEJ) or homology directed repair (HDR). CRISPR uses NHEJ to knockout a gene from an organism, where as HDR is used to insert a gene into an organisms genome. More differences between the two are highlighted here.
The CRISPR technology is now being used in genome editing, and in particular to fight disease. For example, specific gRNA molecules are being specifically designed alongside Cas9 to target antibiotic resistance genes in prokaryotes. Alongside this, labs are trying to modify mosquitos to release into the wild in order to eradicate malaria, however, there are some ethical concerns stipulating this e.g. "designer babies" using genome editing. As a pioneer of the CRISPR/Cas9 technology, Prof. Jennifer Doudna responsibly called for a "global pause" in any clinical application of the CRISPR technology in human embryos to give time to consider the implications of doing so, just as scientists did in the 70s to consider the use of molecular cloning. However, she did say during a TED talks on CRISPR in 2015 that even though genome-engineered humans aren't with us yet, "this is no longer science fiction" and that "in the end, this technology will be used for human genome engineering".
- ↑ Ledford H (3 June 2015). "CRISPR, the disruptor". News Feature. Nature 522 (7554).