Generation and characterisation of a COV434 cell clone carrying a monoallelic FecBB mutation introduced by CRISPR/Cas9

Abstract

The FecBB mutation, which increases the ovulation quota, was identified in a hyperprolific sheep breed, and is associated with a single point mutation (c.A746G; p.Q249R) in bone morphogenetic protein receptor-type 1B (BMPR1B). However, the mechanism of action of the FecBB mutation remains unclear. Here, we describe the application of clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) to introduce the FecBB mutation into the human granulosa cell line COV434 via homology-directed repair (HDR), using single-stranded oligodeoxynucleotides (ssODNs) as template. Upon screening single cell-derived clones, we found one clone containing the FecBB mutation on one allele and knockout of BMPR1B on the other allele, and another clone harbouring an in-frame deletion on one allele and knockout of BMPR1B on the other. These two clones were subjected to functional analysis. We found that the FecBB mutation and knockout of BMPR1B could upregulate the expression of cumulus expansion-related genes, potentially mediated by enhanced activation of the Sma- and Mad-related 1/5 (SMAD1/5) pathway. The FecBB mutation, an in-frame mutation, and knockout of BMPR1B could impair the synthesis of oestradiol in COV434 cells, possibly through inhibition of the SMAD2/3 pathway. Our study provides a useful cell model for further investigation into the mechanism of action of the FecBB mutation.