CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes

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Douglas C et al. Nature Communications 2021 December‍

Major technological breakthroughs have allowed the significant improvement of in vitro models’ physiological relevance,¹ with organoids and other 3D culture methods, for example. However, in vivo studies, and thus the use of laboratory animals, are still very much needed and performed. Legislations are also evolving across the world to more tightly adjust and limit the use of laboratory animals, with the now global “Replacement, Reduction and Refinement” (3Rs) guidelines. A 2010 EU directive sets its ultimate goal to the full replacement of the use of animals for scientific purposes, while improving the welfare of the animals still needed in the meantime.²

One technical shortcoming in the 3Rs guidelines appears in research areas that focus on sex-specific traits or pathologies, such as gametogenesis, placental biology, or sex-specific cancers like prostate cancer. In these cases, research is performed on males or females only. In this context, being able to control offspring sex could significantly reduce the use of laboratory animals. As of today, the means of controlling the sex of offspring are neither effective or humane, as they mostly consist in culling the unwanted animals.

A recent study published in Nature Communications proposed a new genetic system to tackle this issue.³ In their paper, Douglas and colleagues used CRISPR/Cas9 technology to facilitate the generation of single-sex litters. Their system is built on the known lethal phenotype observed in embryos invalidated for the Topoisomerase 1 DNA replication and repair factor. Using H11^Top1 females (expressing a Top1-targeting sgRNA from the H11 neutral locus on an autosome), and males expressing Cas9 from either the X (X^Cas9Y) or the Y (XY^Cas9) chromosome, they showed that co-inheritance of Cas9 and the Top1sgRNA induced sex-specific embryonic lethality with 100% efficiency. Indeed, crossing H11^Top1 females with X^Cas9Y males produced only male descendants, whereas H11^Top1 females mated with XY^Cas9 males only had female offspring. Interestingly, a phenomenon of litter size’s compensation occurred in these cases, as the size of these same-sex litters was superior to 50% of a control litter size. The authors also described that this system could be used to induce sex-specific postnatal phenotypes.

Several novel genetically engineered mouse models were developed for this study, including one designed and generated by genOway, a designer and provider of numerous physiologically relevant preclinical models in multiple research areas.

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References:

1. Horvath P, Aulner N, Bickle M, Davies AM, Nery ED, Ebner D, Montoya MC, Östling P, Pietiäinen V, Price LS, Shorte SL, Turcatti G, von Schantz C, Carragher NO. Screening out irrelevant cell-based models of disease. Nat Rev Drug Discov. 2016 Nov;15(11):751-769.
2. ‍https://ec.europa.eu/environment/chemicals/lab_animals/index_en.htm
3. ‍Douglas C, Maciulyte V, Zohren J, Snell DM, Mahadevaiah SK, Ojarikre OA, Ellis PJI, Turner JMA. CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes. Nat Commun. 2021 Dec 3;12(1):6926.