CRISPR-Cas9 technique in the lab

Precise and efficient genome editing has triggered a revolution in making genetic manipulations possible essentially in all types of cells and organisms. There are many approaches like ZFNs and TALENs have been developed for genome editing. Among which, the most recent and broadly applicable one is CRISPR-Cas9 which is short for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9.

CRISPR-Cas9 is a system adapted by bacteria to fight against invading viruses by keeping the memory of previously infected ones. It’s a two part system: Cas9, an enzyme to cut the DNA at a specific position and a piece of RNA, called guide RNA(gRNA) to direct Cas9 the right point to make a cut in the genome. The complex of these two i.e. (gRNA+ Cas9) keeps bouncing along the genome and when they find a spot where gRNA matches, it inserts between the double strand of the helix, ripping it apart and this triggers the Cas9 protein to cut. In this way the prokaryotes get rid of the invading viruses. But in eukaryotes, there are several pathways available to follow a repair mechanism for the broken DNA. This is the idea behind CRISPR-Cas9 genome editing technology. Now what we got is that, all of a sudden, the cell will be in panic because it got a piece of broken DNA and call upon the possible ways to get it repaired. NHEJ(Non-Homologous End Joining) and HDR( Homology Directed Repair) are the two pathways which will be a help to the cell in such condition. NHEJ simply allows the joining of the two ends of the broken DNA by adding or removing one or two base pairs. This method is suitable to be applied in case of gene knockouts but are not suitable to fulfil the purpose of genome editing. HDR uses the homologous DNA from the chromosome to repair and scientists can hijack this mechanism by designing a false copy of the homologous DNA, keeping the two ends as it is and manipulating the sequence in the middle to fool the cell. By this way, genome editing using CRISPR-Cas9 technique can be performed to remove, add or alter the DNA in a genome.

How it actually works in the lab?? Scientists would first design gRNA complementary to the segment of DNA to be manipulated. Then they inject this gRNA sequence along with the Cas9 or any other nucleases like Cpf1. Cas9+gRNA complex bounces along the genome and attaches every time it comes in contact with a PAM which is 2-3 bp long sequence found adjacent to target DNA. PAM makes the gRNA match search easy and fast and helps it in grabbing and unzipping the double strand. When gRNA finds a match, Cas9 cut the target DNA and once the DNA is cut, scientists use the cell’s own DNA repair machinery to add or remove pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence. They do so by injecting the donor DNA, the new gene together with guide RNA and cas9 with the help of a microscope and a tiny needle or can punch holes in cells with electric currents and let these things just float in, use guns to shoot them in stuck-on tiny bullets, or introduce them encapsulated in bubbles of fat that fuse with the cell membrane and release their contents inside.

References:

1). Doudna, Jennifer A., and Emmanuelle Charpentier. “The new frontier of genome engineering with CRISPR-Cas9.” Science346, no. 6213 (2014): 1258096.

2). Carroll, Dana. “Focus: Genome Editing: Genome Editing: Past, Present, and Future.” The Yale journal of biology and medicine 90, no. 4 (2017): 653.