CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

A long-standing objective of biomedical researchers is the creation of effective and trustworthy techniques for making precise, targeted changes to the genome of living cells. There is a lot of excitement surrounding a new tool based on the Streptococcus pyogenes CRISPR-associated protein-9 nuclease (Cas9). This comes after numerous attempts over the years to change how genes function, including RNA interference and homologous recombination (RNAi). Particularly RNAi became a mainstay in laboratories; enabling low-cost, high-throughput investigation of gene function, but it is limited by the fact that it only provides transient inhibition of gene function and unpredictable off-target effects. Other methods of targeted genome modification, such as transcription-activator like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), allow for the creation of permanent mutations by inducing double strand breaks that activate repair pathways. Since these methods require expensive and time-consuming engineering, they are rarely used, especially for large-scale, high-throughput studies.

Cas9 / CRISPR
Selective bacteria and archaea are able to respond to and get rid of invasive genetic material thanks to the functions of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes. In E. coli, these repetitions were first spotted in the 1980s, but but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus.

CRISPR mechanisms
The invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus between a series of short repeats, one of the mechanisms that has been identified (around 20 bps). The loci are then translated into small RNAs (crRNA, or CRISPR RNA), which are used to direct effector endonucleases that target invasive DNA based on sequence complementarity.

Applications as a Genome-editing and Genome Targeting Tool
The CRISPR/Cas9 system was first demonstrated in 2012 and has since gained widespread use. Important genes in numerous cell lines and organisms, including human, bacteria, zebrafish, plants, yeast, Drosophila, monkeys, rabbits, pigs, rats, and mice, have already been successfully targeted using this technique. This technique has now been used by numerous groups to introduce single point mutations (deletions or insertions) via a single gRNA in a specific target gene. It is also possible to cause large deletions or genomic rearrangements, such as inversions or translocations, using a pair of gRNA-directed Cas9 nucleases as an alternative. The use of the dCas9 variant of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualisation of particular genome loci is a recent and exciting development. To change target specificity with the CRISPR/Cas9 system, the crRNA only needs to be redesigned. In contrast, other genome editing tools like TALENs and zinc fingers call for redesigning the protein-DNA interface. Furthermore, by producing large gRNA libraries for genomic screening, CRISPR/Cas9 enables quick genome-wide interrogation of gene function.

 Future of CRISPR/Cas9
Because of the system's simplicity, high efficiency, and adaptability, Cas9 has advanced remarkably quickly into a set of tools for cell and molecular biology research. The CRISPR/Cas system is by far the most user-friendly designer nuclease system currently available for precise genome engineering. It is now evident that Cas9 is capable of more than just cleaving DNA, and that the extent to which it can be used to recruit proteins to specific genome loci is likely only constrained by our creativity.

By:
Dr. Swati Tyagi
Assistant Professor
School of Biosciences