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New Technique to Boost CRISPR-Cas9 Efficiency

by Julia Samuel on August 30, 2016 at 5:20 PM
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New Technique to Boost CRISPR-Cas9 Efficiency

The efficiency of the ground-breaking gene-editing technology CRISPR-Cas9 can now be boosted by up to five times using a simple technique.

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.

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The new technique could make it easier to create and study knockout cell lines and, potentially, disable a mutant gene as a form of human therapy.

In the study, published in the journal Nature Communications, the researchers described that the process can be made more efficient by 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.
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"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 from the University of California, at Berkeley, in the US.

The technique boosts the efficiency of all CRISPR-Cas9s, even those that initially failed to work at all.

Corn portrays CRISPR-Cas9 gene editing as a competition between cutting and DNA repair.

Once Cas9 cuts, the cell exactly replaces the cut DNA, which Cas9 cuts again, in an endless cycle of cut and repair until the repair enzymes make a mistake and the gene ends up dysfunctional.

Perhaps, he said, the oligonucleotides decrease the fidelity of the repair process, or make the cell switch to a more error-prone repair that allows Cas9 to more readily break the gene.

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.

Because 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. This can make it difficult to knock out all copies at once, and higher efficiency greatly increases the chance of success.

High efficiency also is essential when knocking out genes to correct hereditary mutations in humans.

Physicians have speculated about knocking out genes that make people susceptible to infectious diseases, such as AIDS, or prone to autoimmune, inflammatory or neurodegenerative disorders.

However, it remains to be seen whether the approach described by Corn and colleagues could be used in a therapeutic context.



Source: IANS
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