Scientists have found a new way to boost the efficiency of the gene-editing tool CRISPR-Cas9 by five times in most types of human cells.
This makes it easier to create and study knockout cell lines and potentially disable a mutant gene as a form of human therapy, according researchers from University of California Berkeley (UC Berkeley) in the US.
CRISPR-Cas9 is the go-to technique for knocking out genes in human cell lines to discover what the genes do, but the efficiency with which it disables genes can vary immensely.
Scientists are constantly discovering new genes or the proteins they code for, but it is much harder to figure out their role in the body or in disease.
The key to discovering this role is disabling the gene to see what happens when it is removed.
While CRISPR-Cas9 can accelerate the process of making knockout cell lines, researchers must sometimes make and screen many variations of the genetic scissors to find one that works well.
They found that this process can be made many times more efficient with a simple tweak.
The key is introducing into the cell, along with the CRISPR-Cas9 protein, short pieces of DNA that do not match any DNA sequences in the human genome.
The short pieces of DNA, called oligonucleotides, seem to interfere with the DNA repair mechanisms in the cell to boost the editing performance of even mediocre CRISPR-Cas9s between 2.5 and 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 Jacob Corm from UC Berkeley.
The technique boosts the efficiency of all CRISPR-Cas9s, even those that initially failed to work at all.
With higher efficiency, researchers will have better success at creating the knockouts they want, and then using those knockout cell lines to explore the function of a gene or a group of genes.
Since most long-lived cell lines are derived from cancer cells - including the very popular HeLa cell line - these cell lines typically have more than the normal two copies of each gene, researchers said.
This can make it difficult to knock out all copies at once and higher efficiency greatly increases the chance of success, they said.
The findings were published in the journal Nature Communications.