CRISPR is an abbreviation for ‘clustered regularly interspaced short palindromic repeats’, and is a term used to describe unusual repeated base sequences in bacterial genomes. In 2011, it was known that CRISPRs, along with Cas proteins, function as an adaptive immune system in prokaryotes; that CRISPRs are transcribed into long RNA molecules (pre-crRNA) that are then cleaved within the repeat sequences to yield small CRISPR RNAs (crRNAs); and that the Cas9 nuclease was a multi-domain crRNA-binding protein. However, it was not clear how this biochemical machinery could be redeployed to perform gene editing. Charpentier and co-workers discovered trans-activating crRNA (tracrRNA) — another part of this bacterial immune system — and identified its role in crRNA maturation.
Next, Charpentier, Doudna and co-workers proved that crRNA and tracrRNA were both needed for Cas9 to cleave DNA. They also investigated whether it was possible to control the sequence specificity of the Cas9 nuclease, showing that crRNA and tracrRNA could be combined to form a single RNA molecule that can guide the Cas9 nuclease and direct DNA cleavage to occur at a specific and pre-programmed site. These remarkable discoveries enable very precise editing of a genome. Since these breakthroughs, the CRISPR–Cas9 system has evolved into a powerful tool for altering genomes and can be used to analyse the effect of DNA sequence variations in fundamental science experiments. In a more applied context, efforts to develop new treatments for genetic diseases based on CRISPR–Cas9 gene editing are also ongoing around the world.
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