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

Abstract

Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only litters with one hundred percent efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes. Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.

Fig. 1. Screening of Top1 guides and generation of the X^Top1 mouse.

a Top1 locus showing sgRNA targeting sites. Exon numbers indicated. b Scheme for sgRNA screening. c TOP1 western blot in eGFP only and mCherry - eGFP double-positive mESCs after sgRNA2 transfection (TOP1 = 110 kDa, TUBULIN = 50 kDa). The assay was performed twice in two independent experiments. d Schematic of X^Top1 transgenic locus. e mCherry western blot in X^Top1Y tissues; L liver, K kidney, T testis, B brain, S spleen, wt; C57BL/6 XY liver (TUBULIN = 50 kDa, mCHERRY = 30 kDa). Western blot was repeated three independent times. f Mating strategy. g Offspring from matings between X^Top1Y males and either homozygous R26^Cas9 females (left column) or control wild-type females (right column). n=number of offspring. p-value shows the significance of deviation from 1:1 ratio (Chi-squared test). sgRNA single guide RNA, mESCs mouse embryonic stem cells, FACS fluorescence-activated cell sorting, kDa kilodalton, eGFP enhanced Green Fluorescent Protein, hu human, pCbh hybrid CBA promoter, TOP1 Topoisomerase 1, ctrl control.

Fig. 2. Generating and examining the functionality of the H11^Top1 mouse.

a Schematic of H11^Top1 transgenic locus. b mCherry in vivo imaging showing H11^Top1 mouse (far right). Raw data images provided as a Source Data file. c Mating strategy. d Offspring from matings between H11^Top1/+ males and either homozygous R26^Cas9 females (first two columns) or control wild-type females (right column). n = number of offspring. p-value shows the significance of deviation from 1:1 ratio (Chi-squared test). e Top1 mutation efficiency in E3.5 wild-type (+/+) and H11^Top1/R26^Cas9 embryos (median = 75%). Box plots describe minima, maxima and median with error bars (s.d.). p-value; two-tailed Mann–Whitney test, n = number of embryos). sgRNA single guide RNA, Top1 Topoisomerase 1, pCbh hybrid CBA promoter, ctrl control, E embryonic day.

Fig. 3. Examining the X^Cas9 and Y^Cas9 functionality to generate single-sex litters.

a Schematic of X-linked Cas9-eGFP transgenic locus. b Schematic of the Y-linked Cas9-eGFP transgenic locus. Exon 18 is skipped in wild-type Uty expression. Exons 19-30 are inserted into the transgene as cDNA. HA^L homology arm left, HA^R homology arm right, NLS nuclear localisation signal. c, d Western blot of X^Cas9 and Y^Cas9 tissues. L liver, K kidney, S spleen, T testis. (CAS9 = 158 kDa (arrow), GAPDH = 37 kDa). Western blot has repeated a minimum of three independent times. e Mating strategies. f Sex genotyping of pups born from X^Cas9Y males (left) or XY^Cas9 males (middle) bred with homozygous H11^Top1 females or control X^Cas9Y males (right) mated to wild-type females. n = number of pups. p-value shows the significance of deviation from 1:1 ratio (Chi-squared test). sgRNA single guide RNA, Chr chromosome, pCAG promoter CMV early enhancer/chicken β-actin, eGFP enhanced Green Fluorescence Protein, pPGK promoter phosphoglycerin kinase, Neo Neomycin, NLS nuclear localisation signal, DTA diphtheria toxin A, wt wild-type, ctrl control.

Fig. 4. Transient introduction of an sgRNA targeting Atm generates a sex-specific phenotype.

a Ovary sections from wild-type and Atm mutant immunostained for germ cell marker MVH (magenta). Dotted circles indicate the degenerated ovary (n = 3 wildtype, n = 4 Atm mutant ovaries). Scale bars: 100 μM. b Total and primordial follicle quantification in 8-week-old Atm mutant and wild-type ovaries. Error bars: mean ± s.d. Statistical analysis performed using Mann–Whitney test (two-tailed). c Periodic-acid Schiff-stained wild-type and Atm mutant testis, showing a complete stage IV, midpachytene germ cell arrest (arrows) in the mutant. Scale bars: 200 μM. d Testis weights in wild-type (n = 6) and Atm mutant (n = 6) 8-week-old males. Error bars: mean ± s.d. Statistical analysis performed using Mann–Whitney test (two-tailed). e Pachytene spermatocytes from wild-type and Atm mutant (as in (d)) males immunostained for SYCP3 (green) and γH2AX (magenta). Two independent biological replicates. Scale bars: 10 μM. mut mutant.