Complex deletion and proximal reinsertion of a 150bp regulatory sequence in the mouse Csf1r promoter mediated by CRISPR-Cas9

This paper describes a deletion/reinsertion event encountered in a genome-editing project using CRISPR-Cas9. The objective was to delete a 150bp enhancer region in the mouse Csf1r locus using a pair of guides and a homology-dependent repair (HDR) template. The editing was successful in generating a founder pup with the anticipated precise deletion. However, the deleted fragment and a duplicated copy of part of the HDR template was reinserted around 50bp downstream. The reinsertion event was recognised because the PCR primer site used in genotyping was duplicated, so that there were three PCR products in a heterozygous animal and two in a homozygote. The event we describe is more subtle and more difficult to detect than large-scale rearrangements reported by others. We suggest that any genomic deletion mediated by CRISPR-Cas9 needs to be confirmed by assessing the copy number in the genome.

Reduced tumour incidence in mice with inherited seborrhoeic dermatitis

121 mice homozygous for the gene seb (inherited seborrhoeic dermatitis) and their 142 unaffected heterozygous littermates were kept for their natural lifespan. Heterozygotes showed 84.1% total tumour incidence in males and 95.9% in females. The most common neoplasms were lymphomas, osteomas, lung tumours and neoplasms of the female genital tract. Homozygotes showed a tumour incidence of 36.1 % in males and 45.0% in females. The reduction in incidence included all types of neoplasms except epithelial tumours of the skin: skin tumours were detected in 11 homozygous but only in one heterozygous animal. Life expectancy was not affected significantly by genotype. Homozygous mice showed rough and greasy fur and became alopecic with age. Energy intake was increased but growth and depository fat was reduced compared with heterozygous mice. Higher heat loss may incompletely be compensated by higher metabolic rate and thus 'dietary restriction' results in decreased tumour rates. As females show small gonads and a higher increase in food consumption hormonal factors may also be involved.