158 Effect of a custom-designed transfection buffer on transfection rates and gene editing success in fibroblast cells of buffalo, cattle, goats, and sheep

Electroporation stands as one of the most widely employed methods for introducing exogenous compounds into mammalian cells. The success of electroporation depends upon several critical factors, with the buffer playing a pivotal role. In this preliminary study, we conducted an assessment of the impact of a custom-designed transfection buffer on transfection rates and gene editing outcomes in cells from buffalo, cattle, sheep, and goats. Throughout the experiments, electroporation conditions, comprising cell number, pulse strength, pulse duration, cuvette size, and buffer volume remained the same and three biological replicates were performed with five to seven technical replicates. Following 48 h of electroporation utilising the pACGFP1-N1 plasmid, our results revealed that goat cells exhibited the highest transfection rates (23.63% ± 2.35), followed by buffalo (21.49% ± 2.49), cattle (18.42% ± 1.50), and sheep (15.63% ± 1.20). In addition, apoptosis rate data indicated that buffalo cells exhibited the highest rates (12.51% ± 1.82), followed by cattle (11.47% ± 2.24), goats (10.45% ± 3.44), and sheep (6.981% ± 0.17). After subjecting cells to electroporation with CRISPR sgRNA and Cas9 that targeted the myostatin (MSTN) gene, the analysis using T7E, TIDE, and ICE methods indicated similar gene editing rates (25% to 30%) across the selected species. The single-cell clonal generation experiments indicated that buffalo cells exhibited lower cell attachment (40%) and proliferation rates (70%) in comparison to goat (77% and 75%) and sheep cells (85% and 83%). The generated MSTN-gene-edited cell clones showcased both mono-allelic and bi-allelic editing in cells. Further experiments are required to compare the CRISPR editing rates of commercial transfection buffers with those obtained using our custom-designed transfection buffer. In summary, the results of this preliminary study highlight the feasibility of our custom-developed buffer for gene editing using CRISPR components and offer a promising avenue for gene editing studies in farm animal species.