Scientists at the University of California, Berkeley, have developed a way to increase the efficiency of the increasingly popular CRISPR-Cas9.
CRISPR-Cas9 is a gene editing technique in which the combination of a nuclease and guide RNAs allow for the cutting of a genome at a target location, enabling genes to be knocked out from human cell lines in order to discover what those genes do.
Currently the efficiency with which it disables these genes can hugely vary but, thanks to new research, the process can now operate up to five times more efficiently.
A key component in figuring out the role of genes in the body or disease is finding out what happens when said gene is disabled. CRISPR-Cas9 accelerates the process but can sometimes require scientists to screen several variations in order to find one that works.
However, the new has found that by introducing short pieces of DNA that do not match any DNA sequences in the human genome, alongside the CRISPR-Cas9 protein, into the targeted cell, the efficiency of the process is massively boosted. Notably, this efficiency increase applies to all CRISPR-Cas9s, including those that initially failed to work entirely.
The function of these short DNA pieces, which are called oligonucleotides, is that they interfere with DNA repair mechanisms in the targeted cell; an operation that boosts the performance of CRISPR-Cas9s, even those performing poorly, by a range of 2.5 to 5 times.
“It turns out that if you do something really simple — just feed cells inexpensive synthetic oligonucleotides that have no homology anywhere in the human genome — the rates of editing go up as much as five times,” said lead researcher Jacob Corn, the scientific director of UC Berkeley’s Innovative Genomics Initiative and an assistant adjunct professor of molecular and cell biology.
Corn’s method relied on the suspicion that the unreliability of Cas9’s efficiency stemmed from the mechanism by which DNA is repaired, given that DNA repair mechanisms are different across cells.
The idea of the oligonucleotides came from the reasoning that the introduction of random DNA into the cell could potentially confuse the repair process and thus increase the rate of a successful knockout.
This increased efficiency is hugely important on two fronts.
The first is understanding the role of genes, as a better chance of knockout will aid with the success rate of study. This is particularly prevalent when looking at long-lived cell lines, such as HeLa cells, as such cell lines typically have more than the regular two copies of each gene.
Increased CRISPR efficiency makes it much more likely to successfully knock at out all the copies at once.
Perhaps more importantly, though, is that gene editing is crucial to the combating of currently incurable genetic conditions, allowing for the correction of hereditary mutations.
It is speculated that genes such as those that make people susceptible to infectious diseases like AIDS could be knocked out, though it remains to be seen whether the Berkeley team’s approach is usable in a therapeutic context.