Scientists extend control of CRISPR-Cas9 genetic inheritance in mammals

Almost three years ago, researchers at the University of California at San Diego announced the world’s first approach based on CRISPR-Cas9 gene editing to control heredity in mammals.

The 2019 achievement described research by Hannah Grunwald, then a UC San Diego graduate student, and Associate Professor Kimberly Cooper, who used ‘active genetic’ editing, a technology developed at UC San Diego, to influence gene inheritance in mice. This gives biologists the ability to control which copy of a gene is inherited from generation to generation with potential for a variety of biomedical and environmental applications. The research was successful in female mice but not in males, possibly due to differences in the timing of key events in females and males during the reproductive cell division process known as meiosis.

Researchers found that CRISPR-Cas9 gene editing during meiosis was essential for controlling allelic inheritance in male and female mice. The image shows male germ stem cells (magenta only) and expression of Cas9 in meiotic spermatocytes marked by overlapping magenta and green.

Led by graduate student Alexander Weitzel, Grunwald, Cooper and their colleagues have now successfully developed control of CRISPR-Cas9 inheritance in male mice by moving the gene editing window to more closely match the time of meiosis in both sexes. Their results were published on December 23, 2021 in the journal PLOS Biology.

This achievement advances the prospects of scientists being able to use gene editing for novel laboratory models in a range of research activities, from human disease investigations to the design of therapeutic drugs to elimination of invasive species.

“For these gene conversion strategies to work in any setting – in the lab or in wild populations – you need the gene conversion mechanism to work in both males and females,” said Cooper, professor. Associate in the Cell and Developmental Biology Section, Biological Sciences Division. “It seems the reason this process previously worked in women was because we were closer to the female meiotic window. Now that we’ve moved the Cas9 expression to the meiotic window in men, it works in them too.

As before, the researchers used an active piece of genetic DNA that copies genetic information, known as “CopyCat”, in mice. In the new experiments, the researchers used regulatory DNA from Spo11, a gene known to be involved in meiosis in males and females, to control the expression of the Cas9 protein that cuts DNA.

“Since timing is so important, reaching this sweet spot for meiosis was important for the conversion of male genes,” Cooper said.

As before, the success of the new strategy had certain limitations. In order to land in the male time window, while remaining in the female time window, the gene conversion process became less efficient, possibly due to a lower level of Cas9 expression. Research underway in Cooper’s lab is investigating this problem.

“While showing that CRISPR-Cas9-mediated gene conversion is possible in both male and female mouse germ lines, our work reveals nuances that differ from insects and that need to be considered for further refinement and implementation. in rodents, “note the authors in the article.

At the same time, Grunwald, Weitzel and Cooper expanded their work by publishing a type of instruction manual for other laboratories interested in gene conversion in mammals.

In one Natural protocols The article, also published Dec. 23, describes details of their CRISPR-Cas9 gene conversion system and its potential for use in the laboratory and in wild rodent populations. They outline the technical hurdles that must be overcome before scientists implement such strategies for applications in any context. Expanding this system, the authors write, would increase laboratory efficiency, which has advantages on several levels.

“If implemented successfully in the laboratory, gene conversion mediated by CRISPR-Cas9

promises to save money, time and animal lives, while simultaneously expanding our ability to investigate some of the most complex development issues and the most prevalent human diseases, ”they say in the paper.

Co-authors of the PLoS Biology paper include Alexander Weitzel, Hannah Grunwald, Ceri Weber, Rimma Levina, Valentino Gantz, Stephen Hedrick, Ethan Bier and Kimberly Cooper. Research support for this article was provided by a Packard Fellowship in Science and Engineering (2015-63114), the David and Lucile Packard Foundation, National Institutes of Health (R21GM129448 and R01AI131081), an Allen Frontiers Group Distinguished Investigators Award , a donation from the Tata Trusts in India to the TIGS-UC San Diego and TIGS-India and NIH Training Fellowships (T32GM007240 and T32GM133351).

the Natural protocols Perspective was supported by Tata Trusts India at TIGS-UC San Diego and TIGS-India and by a training grant from NIH (T32GM007240).

Notes on Competing Interests: Gantz, Bier, Hedrick (co-founder, with participation) and Cooper (participation) hold advisory board positions with Synbal Inc., which could potentially benefit from the research results. Bier also owns a stake in Agragene, Inc. and sits on the board of directors of Synbal.


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