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

Autor: Jose B; Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Punetha M; Division of Animal Physiology and Reproduction, ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, 125001, India. Electronic address: meeti.punetha@icar.gov.in., Tripathi MK; Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Khanna S; Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Yadav V; Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Singh AK; Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Kumar B; Division of Animal Reproduction, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Singh K; Division of Surgery, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Chouhan VS; Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India., Sarkar M; Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, 243122, India. Electronic address: mihir.sarkar@icar.gov.in.
Jazyk: angličtina
Zdroj: Theriogenology [Theriogenology] 2023 Nov; Vol. 211, pp. 125-133. Date of Electronic Publication: 2023 Aug 18.
DOI: 10.1016/j.theriogenology.2023.08.008
Abstrakt: 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.
(Copyright © 2023 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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