Meiotic Cas9 expression mediates genotype conversion in the male and female mouse germline

Abstract

Highly efficient genotype conversion systems have potential to facilitate the study of complex genetic traits using laboratory mice and to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of genotype conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double strand DNA break is feasible in the female mouse germline. In the male germline, however, all double strand breaks were instead repaired by end joining mechanisms to form an 'insertion/deletion' (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate genotype conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient double strand DNA break formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of genotype conversion and further increase the rate of 'super-Mendelian' inheritance from both male and female mice.

High-resolution in situ analysis of Cas9 germline transcript distributions in gene-drive Anopheles mosquitoes

“…Given its tight transcript localization in the oocyte where germ cells form, oskar might prove effective in restricting Cas9 activity to meiotic components of the germline in both genders, mirroring the developmental timing in testes to premeiotic stem cells. We also observed tight meiotic localization of spo11 , which in mammals has been used to control Cas9 expression, resulting in drive through both males ( Weitzel et al 2021 ) and females ( Grunwald et al 2019 ). In mosquitoes, spo11 transcripts are not present prior to or during the arrest phase, but express and accumulate in the oocyte following a blood meal when transcription restarts.…”

A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila

“…Indeed, a nickase gene-drive system could be tested in Anopheles, or other species such as Aedes or Culex mosquitoes that present a genome three times bigger than Anopheles (Main et al, 2021;Severson et al, 2004), and where off-target effects may be more pervasive due to genome size. Furthermore, gene drives also demonstrated their ability to bias Mendelian inheritance using mice (Grunwald et al, 2019;Weitzel et al), and a nickase-based gene-drive system should be portable to these animals to explore potential benefits to reduce off-target effects observed when editing mammalian embryos (Aryal et al, 2018). Altogether, our proof-of-principle study represents the first step towards the development of next-generation nickase-based gene-drive designs bringing potential advantages to future applications of these technologies.…”

A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila

“…Indeed, a nickase gene-drive system could be tested in Anopheles and species with much larger genomes, such as Aedes or Culex mosquitoes ( Main et al, 2021 ; Severson et al, 2004 ), to study the pervasiveness of off-target effects across genome sizes and in the wild. Furthermore, gene-drives can bias Mendelian inheritance in mice ( Grunwald et al, 2019 ; Weitzel et al, 2021 ). In the future, a nickase-based gene-drive system could also be applied to mice or to reduce off-target effects when editing mammalian embryos ( Aryal et al, 2018 ).…”