Disruption of the sheep BMPR-IB gene by CRISPR/Cas9 in in vitro-produced embryos

Abstract

BMPR-IB (also known as FecB) is a key candidate gene for the genetic control of sheep reproductive performance. Loss-of-function mutations in the sheep BMPR-IB gene lead to an increase in ovulation rate and consequently larger litter size. However, the BMPR-IB gene has been identified in only a few sheep breeds. To improve sheep reproduction through modification of the BMPR-IB gene, we designed an sgRNA to target the sheep BMPR-IB gene by using the CRISPR/Cas9 system. First, we performed gene editing by injecting Cas9/sgRNA into the cytoplasm of one-cell fertilized eggs. A total of 88 embryos were assayed by T7EI digestion and Sanger sequencing. The results reported that the efficiency of gene modification was 37.5% (33/88) and increased with the developmental stage of embryo from the 2-cell stage to the blastocyst stage. Of the 33 gene editing embryos, 12 (36%, 12/33) were homozygous and 21 (64%, 21/33) were heterozygous. Moreover, sequence analysis of the PCR products from the positive embryos revealed that there were more than 10 modification forms that resulted in frame shift and truncated proteins. Further analysis by cloning and sequencing of each individual embryo showed a high level of mosaicism. In addition, off-target event analysis revealed that none of the off-target mutations was introduced into the embryos. Our results indicated that the Cas9/sgRNA system is a simple and efficient tool that may potentially be used in the genetic modification of the sheep BMPR-IB gene in vivo.