CRISPR/Cas mediated disruption of BMPR-1B gene and introduction of FecB mutation into the Caprine embryos using Easi-CRISPR strategy

• Published in: Theriogenology 2023-11, Vol.211, p.125-133
• Main Authors: Jose, Bosco, Punetha, Meeti, Tripathi, Manoj Kumar, Khanna, Shivani, Yadav, Vijay, Singh, Amit Kumar, Kumar, Brijesh, Singh, Kiranjeet, Chouhan, Vikrant Singh, Sarkar, Mihir
• Format: Article
• Language: English
• Subjects: BMPR-1B; Caprine; CRISPR/Cas; Embryo; Ribonucleoprotein complex; ssODN
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2023.08.008

Bone Morphogenetic Proteins play a significant role in ovarian physiology and contribute to the reproductive fitness of mammals. The BMPR-1B/FecB mutation, a loss of function mutation increases litter size by 1–2 with each number of mutated alleles in sheep. Considering demand-supply gap of the meat industry, and low replacement rate of indigenous caprine species, the conservative BMPR-1B locus can be explored, and FecB mutated goats can be produced. The experiment one produced CRISPR/Cas mediated KO transferable caprine embryos, and experiment two generated caprine embryos with desired FecB mutation using Easi-CRISPR strategy. In the KO experiment, Cas9 and BMPR-1B guide RNA (100:100ng/ul) were electroporated into single stage caprine zygotes at 750V, 10 ms and 1pulse using Neon transfection system. In the second experiment, phosphorothioate (PS) modified single-stranded oligodeoxynucleotide (ssODN) was used as an HDR template along with CRISPR components (100:100ng/ul, ssODN 100ng/ul). The precise time and method of electroporation, RNP format of CRISPR components and PS modified asymmetric ssODN were the factors that affected the production of mosaicism free BMPR-1B edited caprine embryos. The editing efficiency of KO and KI experiments was 68.52 and 63.16% respectively, and successful production of goats with higher mean ovulation rate can be realized with addition of embryo transfer technology to these experiments. •FecB mutation increases litter size by 1–2 extra lambs.•A Single Nucleotide Polymorphism (SNP) at position 746(A to G) of BMPR-1B locus causes loss of function of BMPR-1B resulting in increased prolificacy.•We optimized electroporation conditions to deliver Cas9: sgRNA RNPs to caprine zygotes to introduce gene silencing mutations.•Easi-CRISPR strategy for the KI experiment were adopted to avoid mosaicism in embryos and enhance HDR efficiency.