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

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) 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 gene 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 DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.

SPEN is required for Xist upregulation during initiation of X chromosome inactivation

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.

Balancing memory fidelity and representational stability in the female mouse accessory olfactory bulb

Sensory systems must balance the value of efficient coding schemes against the need to update specific memorized representations without perturbing other memories. Here we describe a unique solution to this challenge that is implemented by the vomeronasal system (VNS) to encode and remember multiple conspecific individuals as part of the Bruce Effect (BE). In the BE, exposure of a pregnant female mouse to the odors of an unfamiliar male leads to failure of the pregnancy (pregnancy block) via the VNS. Following mating and sensory exposure, however, the female becomes protected from a pregnancy block by the stud individual. While this form of natural learning has been proposed to depend on changes in the representation of his odors in her accessory olfactory bulb (AOB), a key VNS structure, there are no direct comparisons of in vivo sensory responses before and after imprinting. It has further been suggested that these changes simply render the AOB insensitive to stud odors. However, the combinatorial odor code used by the AOB and the significant overlap in the odor composition of different males means that silencing responses to one individual is likely to degrade responses to others, posing potential problems for more general sensory encoding. To identify the neuronal correlates of learning in the context of the BE, we recorded extracellular responses of AOB neurons in vivo in mated and unmated female mice upon controlled presentation of urinary chemosignals, including urine from both the stud and males of a distinct strain. We find that while initial sensory responses in the AOB (within a timescale required to guide social interactions) remain stable, responses to extended stimulation (as required for eliciting the pregnancy block) display selective attenuation of stud-responsive neurons. Based on our results, we propose a model that reconciles the formation of strong, selective memories with the need to sustain robust representational bandwidth by noting a distinction between the representations of brief and extended stimuli. This temporal disassociation allows attenuation of slow-acting endocrine processes in a stimulus-specific manner, without compromising consistent ongoing representations of stimuli that guide behavior.