# Design, Screening, and Impact of sgRNAs Targeting Bovine Prolactin Gene Receptor on Embryonic Development Using Stably Transfected Cell Lines

## TL;DR

This study designed and tested three gene-editing tools targeting a hormone receptor in cattle embryos, finding that one tool significantly affected embryo development.

## Contribution

The study introduces specific sgRNAs targeting the bovine prolactin receptor gene and demonstrates their impact on embryonic development using stable cell lines.

## Key findings

- sgRNA139 showed the highest DNA cleavage and repair efficiency.
- sgRNA109 significantly reduced embryonic cleavage and blastocyst rates.
- Stable edited cell lines can regulate later stages of embryonic development.

## Abstract

In this study, three specific sgRNAs (sgRNA139, sgRNA128, and sgRNA109) targeting exon 9 of the prolactin gene receptor (PRLR) in fetal cattle were designed. DNA cleavage was mediated using the CRISPR/Cas9 system, and stable cell lines were screened. Through somatic cell nuclear transfer technology, the effects of different editing sites on embryonic development were systematically investigated. The results showed that sgRNA139 exhibited the highest DNA cleavage and repair efficiency. In terms of embryonic development indicators, sgRNA109 significantly reduced the cleavage rate and blastocyst rate (p < 0.01), while sgRNA139 had no significant effect on the cleavage rate (p > 0.05). These findings confirm that highly specific sgRNAs and their stable edited cell lines, as donor cells, can significantly regulate the later stages of embryonic development. This study provides important theoretical foundations and practical guidance for gene editing and molecular breeding in dairy cattle.

This study designed three sgRNAs (sgRNA139, sgRNA128, and sgRNA109) targeting the prolactin gene receptor (PRLR) in fetal cattle, utilized Cas9 to cleave endogenous DNA, and screened stable cell lines for somatic cell nuclear transfer experiments to investigate the impact of different editing sites on embryonic development. The results showed that sgRNA139 had the highest cleavage efficiency (Fcut = 0.65, Indels = 42.19%), while sgRNA109 had the lowest (Fcut = 0.45, Indels = 35.31%). No significant differences were observed in cell growth status after electroporation (p > 0.05), and the transfection efficiency exceeded 90% after five days of culture. In the evaluation of key embryonic development indicators, sgRNA109 significantly reduced the cleavage rate and blastocyst rate (p < 0.01), whereas sgRNA139 showed no significant effect on the cleavage rate (p > 0.05), but its blastocyst rate was slightly lower than that of the control group (p > 0.05). This study demonstrates that highly specific sgRNAs and stable edited cell lines used as donor cells can significantly regulate the later stages of embryonic development. This study not only provides new experimental evidence for the functional study of the PRLR but also lays an important theoretical foundation for the innovation of molecular breeding technologies in dairy cattle.