Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation re-mains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleo-protein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/mL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/mL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 +/- 3.33%). Surprisingly, we found approximately 95% edited oocytes presented mono-allelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (proto-spacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and addi-tional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjec-tion was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.(c) 2022 Elsevier Inc. All rights reserved.

Automated summary

In this study, the researchers successfully edited the BMP15 gene by injecting CRISPR ctRNP into porcine oocytes, revealing the role of BMP15 in the in vitro maturation of oocytes. Editing BMP15 resulted in reduced BMP15 protein expression, leading to the attenuation of SMAD1/5/8 signaling activation and impaired cumulus expansion in porcine cumulus cell-oocyte complexes (COCs).