Generating single-sex litters: development of CRISPR-Cas9 genetic tools to produce all-male offspring

Charlotte Douglas, Valdone Maciulyte, Jasmin Zohren, Daniel M. Snell, Obah A. Ojarikre, Peter J.I. Ellis, James M.A. Turner

Preprint posted on 7 September 2020 https://www.biorxiv.org/content/10.1101/2020.09.07.285536v1

Say no to the sex reveal party! Generating all-male offspring using sex specific CRISPR-Cas9 genetic tools

Selected by Martin Estermann

Categories: cell biology, developmental biology, genetics, genomics, molecular biology

Background:

Chromosomal sex is established when the gametes fuse at fertilization. In most mammals, oocytes typically carry a single X sex chromosome, whereas the sperm can carry an X or a Y chromosome, resulting in XX (female) or XY (male) embryos. In research there is a preference towards one sex over the other, resulting in the utilization of only half of all the mice born. One of the most evident examples is the study of the reproductive organs. Only male or only female mice are used when studying testicular or ovarian function respectively. In addition, there is a sex-specific demand in the agriculture industry, evident in the need for females in the dairy and egg industry or males in the meat industry.

The generation of the single sex (all male or all female) litters is the solution to these unequal sex requirements. This is also in line with the 3Rs guidelines (Reduction, Replacement and Refinement) in research, that promotes efficient animal use. The authors, taking advantage of differential sex chromosome inheritance and the CRISPR-Cas9 technology, were able to generate all-male litters by inducing specific embryonic lethality in all the female (XX) embryos. This was achieved by the sex specific knock-out of the housekeeping gene topoisomerase 1 (Top1), a key gene in DNA replication and repair. This is the first report of producing all-male litters in the mouse by sex specific CRISPR-Cas9 genetic methods.

Key findings

1) Top1 knock-out is embryonic lethal

A transgenic mouse was generated by integrating a cassette (Fig. 1A), containing a guide against Top1(sgRNA^Top1) gene and a red fluorescent protein reporter (mCherry), into chromosome 11. mCherry expression was detected in preimplantation embryos (Fig. 1B) as well as adult mice, suggesting the integrated transgene is functional. In addition, this transgenic did not induce embryonic lethality. In order to test the effect of the Top1 knock-out, these heterozygous transgenic (sgRNA^Top1/+) mice were bred with a homozygous female mouse expressing Cas9 on chromosome 6 (Rosa26-Cas9) (exp) or with wildtype females (ctrl) (Fig. 1C). In embryonic day (E) 3.5 blastocysts, the ratio of Cas9/sgRNA^Top1 embryos to Cas9/+ was 1:1, consistent with the expected Mendelian ratio. However, at E11.5 and at birth 100% of the embryos were Cas9/+ (Fig 1D), suggesting that Top1 mutation causes embryonic lethality between E3.5 and E11.5.

Fig. 1. Top1 knock out is embryonic lethal. (A) Schematic structure of the sgRNA^Top1 knock-in locus. (B) brightfield and fluorescence imaging of a sgRNA^Top1 E3.5 embryo. (C) Schematic of the mating strategies. Male sgRNA^Top1/+ were crossed with wildtype or homozygous Cas9 (Rosa26-Cas9) females. (D) Offspring genotypes during development from the two different crosses. (Preprint Fig. 2A and 2E-G).

2) Co-inheritance of an X-linked Cas9 and autosomal sgRNA^Top1 causes female lethality

A genetic cassette containing Cas9 and GFP was inserted in the X-linked permissive Hrpt locus to generate an X-linked Cas9 mouse transgenic line (Fig. 2A). X-Cas9Y transgenic males were viable and fertile, confirming that the insertion in the X chromosome was not lethal. To asses if single sex litters can be generated using this system, X-Cas9Y hemizygous males were mated with either wildtype (ctrl) or homozygous sgRNA^Top1females (exp) (Fig. 2B). Equal proportions of male and female pups were recovered from control matings. In contrast, 99% of the pups were male in the experimental cross (Fig 2C). This indicates that the co-inheritance of the X-linked Cas9 and sgRNA^Top1induces lethality in all XX females. Interestingly, although significantly reduced, the litter size was 61% of the control ones, in contrast with the 50% expected (Fig. 2D). This reveals the existence of an in utero compensatory mechanism to maximize the embryo number per litter.

Fig. 2. Co-inheritance of an X-linked Cas9 and autosomal sgRNA^Top1 causes female lethality. (A) Schematic of X-Cas9 knock-in strategy. (B) Schematic of the mating strategies. Male X-Cas9Y were crossed with wildtype or homozygous sgRNA^Top1females. (C) Sex genotyping of pups born from control or experimental matings. (D) Number of pups born per litter in the control and experimental crosses. (Preprint Fig. 3A and 3E-G).

Why I choose this paper:

As a person that works in gonadal development and differentiation, I can completely relate to the problem this research is trying to solve. It is not only time consuming but also cost ineffective to blindly collect tissues knowing that half of them won’t be used due to being of the unwanted sex. This technology provides a great strategy to reduce the inefficient use of animals, one of the key points in research animal ethics. I can see the potential of this technique in the future, and I hope this can be easily translated to other animal research models. In addition, I believe that this research will have an impact on industry, especially in meat production where males are the preferred sex.

Future directions / questions for the authors:

Your data suggest that the embryos die between E3.5 and E11.5. Do you know if they die before, during or after implantation? Were you able to detect any empty implantation sites?
It is really interesting to see an increment in the number of pups born, compared to the expected in control crosses. Do you think this compensatory superovulation will significantly reduce the ovarian reserve over time, resulting in a reduced reproductive life?

Tags: development, embryo, male, sex, transgenic

Posted on: 7 October 2020 , updated on: 12 October 2020

doi: https://doi.org/10.1242/prelights.25102

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Author's response

Peter Ellis shared

Q: Your data suggest that the embryos die between E3.5 and E11.5. Do you know if they die before, during or after implantation? Were you able to detect any empty implantation sites?

Q: It is really interesting to see an increment in the number of pups born, compared to the expected in control crosses. Do you think this compensatory superovulation will significantly reduce the ovarian reserve over time, resulting in a reduced reproductive life?

A: The sex selection system implemented here operates after fertilisation, so there is no way it can alter the number of eggs that were ovulated several days previously. There should therefore be no effect on the mother’s reproductive lifespan. Rather, it is likely that female mice always release more eggs than they have “room” for in the uterus, allowing them to compensate for the loss of some of them prior to implantation.

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Generating single-sex litters: development of CRISPR-Cas9 genetic tools to produce all-male offspring

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