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.
Highlights
Animals are essential genetic tools in scientific research and global resources in agriculture
For the sgRNA component of our system, we needed to target a gene whose disruption would cause early embryo death, thereby eliminating the chance that mutant offspring would survive to term
We cloned guides targeting the Top[1] start codon or DNA-binding domain into an mCherry reporter-expressing vector. This vector was transfected into male mouse embryonic stem cells that constitutively express Cas[9] and an eGFP reporter from the autosomal Gt(ROSA)26Sor locus (R26Cas[9]; Fig. 1b)27. 48 h after transfection, mCherry-eGFP double-positive cells versus eGFPonly control cells were sorted by Fluorescence-activated cell sorting (FACS) (Fig. S1A), and the presence of Top[1] mutations was evaluated (Fig. S1B). sgRNA2 had the greatest mutation efficiency, with 52% of observed sequences exhibiting Top[1] mutations, compared with 22% for sgRNA1 and 29% for sgRNA3
Summary
Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas[9] strategy that produces male- or female-only litters with one hundred percent efficiency. Applications that require females (XX) or males (XY) are especially contentious, because the unrequired sex is generated needlessly and must be culled. The “Replacement, Reduction and Refinement” (3Rs) global guidelines[1] promote efficient animal use, but shortcomings remain. The dairy industry requires females, and as a result, each year around 95,000 male calves are culled in the UK6, 200,000 in Germany[7] and 500,000 in Australia[8]. Flow sorting of X- and Y-bearing sperm is employed to an extent in cattle, albeit with an associated reduction in fertility[10,11]
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