Faster, more efficient CRISPR editing in mice

May 27, 2016

University of California, Berkeley scientists have developed a quicker and more efficient method to alter the genes of mice with CRISPR-Cas9, simplifying a procedure growing in popularity because of the ease of using the new gene-editing tool.

While CRISPR-Cas9 has drawn worldwide attention because of its potential to correct simple hereditary diseases in humans, basic researchers are excited about its 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 - in particular, mice - and see what goes awry. Today's gold standard for creating these "knockouts" or "knockins" 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, called CRISPR-EZ (CRISPR RNP electroporation of zygotes), makes genome editing in mouse embryos even easier.

"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 lead researcher Lin He, a UC Berkeley associate professor of molecular and cell biology. "But it is a major committment to make a novel knockout with genome engineering. 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."

The new method, described in this week's issue of the Journal of Biological Chemistry, 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, allowing them to insert CRISPR-Cas9 gene-editing molecules into embryos with nearly 100 percent success. Electroporation uses a jolt of electricity to create holes in the embryos through which molecules can enter.

Using CRISPR-EZ in a pilot experiment, He's team 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. CRISPR-EZ is a simple and cost-effective methodology, and can be performed on many embryos at once and takes only miliseconds, He said.

"In the not too distant past, it would cost at least $25,000 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. These technical innovations make the mouse an even more powerful tool for modeling human diseases."

The UC Berkeley group is now working with several transgenic mouse facilities in hopes that they will adopt and improve this electroporation technique, which she suspects will also simplify the creation of other transgenic mammals.

Knockouts require IVF team

Creating transgenic mice requires a skilled in vitro fertilization team. Technicians use hormone injections to prepare the females for mating, after which they harvest the fertilized eggs and, before the eggs start to divide, inject them one at a time using a fine needle. Currently, technicians inject two RNA molecules - messenger RNA (mRNA), which codes for the Cas9 protein, and guide RNA, which provides the address for CRISPR-Cas9's target - and hope that the mRNA is properly translated into Cas9 protein and that the protein correctly combines with guide RNA.

The engineered embryos are implanted into a falsely pregnant mouse, where they gestate for about 20 days before birth. Given the inevitable embryo deaths during injection and the failure of the embryos to implant or go to term, the live-birth rate is low, He said.

"The actual percentage of live births from injected embryos is around 10 to 15 percent for most transgenic facilities, which is a problem with the procedure," she said. "Sometimes people collect more than 100 embryos just to generate one or two mice with desirable gene editing."

Electroporation appears to do less damage to the embryos than microinjection: between 30 and 50 percent of the embryos resulted in live births.

Apparently, too, inserting pre-assembled CRISPR-Cas9 molecules into the fertilized egg is a more effective way to edit genes than injecting two molecules - the mRNA and guide RNA - and hoping that they properly self-assemble. Of the live births, 88 percent of the mice had both copies of the target gene edited - a higher success rate than usual for transgenic procedures, He said.

For a more complex procedure - modifying a short DNA sequence within a gene - the method was successful 42 percent of the time in a pilot experiment.

"With such a high success rate, you can use this to test your guide RNAs very quickly," she said. "If your CRISPR-EZ doesn't work, it's not because of delivery; it's likely because your guide RNA design needs to be improved."

The high embryo viability and very high gene-editing efficiency mean researchers need to use fewer mice and can conduct several transgenic experiments simultaneously.

Accidental discovery

In her lab, He studies small bits of RNA called microRNA, which modify how DNA is transcribed and thus control important processes inside a cell. Some types of cancer have been linked to problems with miRNA.

Looking for a simpler way to create transgenic mice to study these regulatory processes, postdoctoral fellow Andrew Modzelewski tested electroporation to see if he could get eggs to more easily take up the Cas9 mRNA and the guide RNA. Despite other researchers' apparent success with mRNA, his initial attempts were unsuccessful. One evening, finding himself out of mRNA and not wanting to waste the prepared embryos, he borrowed CRISPR-Cas9 protein from a neighboring lab, assembled RNP complexes and electoporated them instead.

"It has worked like magic ever since," He said. "You would never think that this would work, because Cas9 is a gigantic molecule. I was surprised that such a huge protein could be electroporated efficiently."

While most universities have transgenic labs where microinjections are performed, electroporation simplifies the procedure enough that individual labs might eventually do it themselves, she predicted.
-end-
Graduate student Sean Chen and postdoctoral fellow Andrew Modzelewski spearheaded this study, Other co-authors of the paper are undergraduate Benjamin Lee and Angus Yiu-Fai Lee, director of the Gene Targeting Facility in UC Berkeley's Cancer Research Laboratory. This work has also benefited from input from Jacob Corn, Mark DeWitt and Jennifer Doudna from UC Berkeley's Innovative Genomics Initiative.

This work was supported by the National Cancer Institute (R01 CA139067) and National Institute of General Medical Science (RO1 1R01GM114414) of the National Institutes of Health.

University of California - Berkeley

Related Cancer Articles from Brightsurf:

New blood cancer treatment works by selectively interfering with cancer cell signalling
University of Alberta scientists have identified the mechanism of action behind a new type of precision cancer drug for blood cancers that is set for human trials, according to research published in Nature Communications.

UCI researchers uncover cancer cell vulnerabilities; may lead to better cancer therapies
A new University of California, Irvine-led study reveals a protein responsible for genetic changes resulting in a variety of cancers, may also be the key to more effective, targeted cancer therapy.

Breast cancer treatment costs highest among young women with metastic cancer
In a fight for their lives, young women, age 18-44, spend double the amount of older women to survive metastatic breast cancer, according to a large statewide study by the University of North Carolina at Chapel Hill.

Cancer mortality continues steady decline, driven by progress against lung cancer
The cancer death rate declined by 29% from 1991 to 2017, including a 2.2% drop from 2016 to 2017, the largest single-year drop in cancer mortality ever reported.

Stress in cervical cancer patients associated with higher risk of cancer-specific mortality
Psychological stress was associated with a higher risk of cancer-specific mortality in women diagnosed with cervical cancer.

Cancer-sniffing dogs 97% accurate in identifying lung cancer, according to study in JAOA
The next step will be to further fractionate the samples based on chemical and physical properties, presenting them back to the dogs until the specific biomarkers for each cancer are identified.

Moffitt Cancer Center researchers identify one way T cell function may fail in cancer
Moffitt Cancer Center researchers have discovered a mechanism by which one type of immune cell, CD8+ T cells, can become dysfunctional, impeding its ability to seek and kill cancer cells.

More cancer survivors, fewer cancer specialists point to challenge in meeting care needs
An aging population, a growing number of cancer survivors, and a projected shortage of cancer care providers will result in a challenge in delivering the care for cancer survivors in the United States if systemic changes are not made.

New cancer vaccine platform a potential tool for efficacious targeted cancer therapy
Researchers at the University of Helsinki have discovered a solution in the form of a cancer vaccine platform for improving the efficacy of oncolytic viruses used in cancer treatment.

American Cancer Society outlines blueprint for cancer control in the 21st century
The American Cancer Society is outlining its vision for cancer control in the decades ahead in a series of articles that forms the basis of a national cancer control plan.

Read More: Cancer News and Cancer Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.