Vol. 10, Issue 7 (2021)

Author(s):
Manoj Kumar Tripathi, Bosco Jose, Shivani Khanna, Pranay Kumar Konda, Anjali, Vikrant Singh Chauhan, Mihir Sarkar and Gyanendra Singh

Abstract:
BMP15, an oocyte derived protein, is a potent stimulator of granulosa cell mitosis. Site specific mutations of BMP 15 gene leads to increase in ovulation rate and litter size in sheep. Such mutations in BMP15 gene has not been observed in caprine species. Genetic control of fecundity is a key area needs to be addressed in caprine species. As a part of study of introduction of FecX^Gr mutation in BMP15 gene in caprine zygotes to increase the fecundity, a prevalidation of knockout of BMP 15 gene was carried out in granulosa cells using CRISPR-Cas gene editing technology. In the present study CRISPR-Cas9 was used to knockout FecX^Gr locus of BMP 15 gene in granulosa cells of goat. Granulosa cells were cultured, FecX^Gr specific guide was designed. PCR amplification of the DNA template containing the sgRNA encoding sequence under the control of aT7 promotor was done which was followed by in vitro transcription to synthesize sgRNA. CRISPR components in the RNP (ribonucleoprotein) format was delivered via electroporation for knocking out of the desired gene in the cultured granulosa cells which was later confirmed by T7Endonuclease test.