Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes

Simple Summary Genome editing is a well-known method for introducing targeted genetic alterations into livestock genomes. These changes must be transferable in the germline in order to be effective in animal breeding. Conventional methods of delivering CRISPR-Cas9 components, such as microinjection in the zygote or editing somatic cells followed by somatic cell nuclear transfer (SCNT), have demonstrated success in various species, including mice and certain domestic animals. However, these methods are often labour-intensive, technically demanding, and associated with variable efficiencies. Electroporation is a more recently described way of delivering Cas9 and sgRNAs into zygotes since it requires less expensive equipment than microinjection and takes less time. In the present study, we have developed an efficient method called CRISPR RNP electroporation of zygote (CRISPR-EP) to reduce mosaicism rates and increase biallelic mutations in buffalo. The developed easy and straightforward protocol for gene editing could serve as a useful method for studying the functional genomics of the buffalo embryos.Abstract Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p <= 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.