Berkeley researchers devise faster, more efficient gene editing in mice

Researchers at University of California, Berkeley, have developed a quicker and more efficient method to alter the genes of mice, simplifying a procedure growing in popularity.

The procedure, or gene-editing tool, known as clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9), has drawn worldwide attention because of its potential to correct simple hereditary diseases in humans.

Basic researchers are excited about the tool's ability to help them understand the causes and develop treatments for more complex diseases, including cancer and dementia. To do that, they need to knock out or modify specific genes in lab animals and see what goes awry.

The current gold standard for creating "knockouts" is to edit the genes inside mouse embryonic stem cells, use these cells to create mosaic mice, and then crossbreed the mice to get a pure genetic strain. Because of CRISPR-Cas9's ability to precisely alter or replace genes, the editing is increasingly being done directly in the fertilized egg, or early embryo.

The new method, described in a paper in the Journal of Biological Chemistry, is called CRISPR-EZ, or CRISPR ribonucleoprotein electroporation of zygotes, makes genome editing in mouse embryos even easier.

It gets around a time-consuming bottleneck in creating knockout mice: using microscopic needles to inject gene-editing molecules into a fertilized egg.

The UC Berkeley researchers found that a simple lab technique called electroporation works much better by using a jolt of electricity to create holes, allowing them to insert CRISPR-Cas9 gene-editing molecules into embryos with nearly 100 percent success.

Using CRISPR-EZ in a pilot experiment, a team led by Lin He, an associate professor of molecular and cell biology, successfully disrupted both copies of a target gene in 88 percent of the mice. The procedure generated a much greater number of edited mice compared to CRISPR microinjection, largely due to a significant improvement in embryo viability.

It is a simple and cost-effective methodology, and can be performed on many embryos at once and takes only milliseconds, He said.

"The key fundamental insights about the biological significance of a gene usually come from in vivo gene-editing studies, in which you generate mice with an altered gene," said He. "I think this technology could greatly reduce the technical barrier for this type of effort and will allow people to focus more on the science rather than be consumed by the process of genetically engineering mice."

"In the not too distant past, it would cost at least 25,000 U.S. dollars and take at least 6 months to make a knockout mouse," said Russell Vance, a UC Berkeley professor of molecular and cell biology and director of the Cancer Research Laboratory, where the transgenic mouse work was performed. "With CRISPR, and improvements such as CRISPR-EZ, the costs and time have both dropped at least 10-fold."

Vance believes that these technical innovations make the mouse "an even more powerful tool for modeling human diseases." Endit