204 Efficient editing of porcine parthenogenetic zygotes by electroporation of Cas9 ribonucleoprotein complexes

Abstract

Gene editing by microinjection is an efficient system to produce mutant livestock; however, microinjection is time-consuming and requires special skill, limiting its use for large-scale production of gene-edited animals. Therefore, the aim of this study was to develop a system to deliver guide (g)RNA/Cas9/ribonucleoprotein (RNP) by electroporation into parthenogenic porcine zygotes. For experiment 1, we delivered gRNA/Cas9 RNP (250 ng μL⁻¹ of each), targeting GATA4 using 2 electroporation conditions. Group 1 (n = 130): 20V, 3 ms, ×2 pulses, 1 repeat; group 2 (n = 102): 20V, 1 ms, ×2 pulses, 2 repeats; and Control (n = 96): parthenogenic zygotes, no electroporation. For experiment 2, we delivered gRNA/Cas9 RNP (250 ng μL⁻¹ of each) targeting ROSA26 by electroporation with 4 conditions compared with delivery of RNP by microinjection: group 1 (n = 17): 20V, 3 ms, ×1 pulses, 1 repeat; group 2 (n = 49): 20V, 3 ms, ×3 pulses, 1 repeat; group 3 (n = 64): 30V, 3 ms, ×1 pulses, 1 repeat; group 4 (n = 61): 30V, 3 ms, ×3 pulses, 1 repeat; group 5 (n = 120): zygotes microinjected with Cas9/ROSA26 sgRNA (25/25 ng μL⁻¹), and Control (n = 76): parthenogenic zygotes, no electroporation. The electroporated zygotes were cultured in porcine zygote medium-3 (PZM-3) with controlled atmosphere, and development was evaluated on Day 2 (cleavage) and Day 7 (blastocyst rate). Gene editing was evaluated on embryos (blastocyst and morulas) by PCR and Sanger sequencing of amplicons including the RNP target site. Data were compared using chi-squared test, and differences were considered significant at P < 0.05. Cleavage rates in experiment 1 were similar for the control (86/96; 89.5%), group 1 (94/102; 92.1%), and group 2 (119/130; 91.5%). Blastocyst rates were higher for the control (46/96; 47%) than for the other groups (P < 0.01). However, for the treated groups, the blastocyst rates were similar, group 1 (19/102; 9.2%) and group 2 (12/130; 18.6%). Furthermore, the non-homologous end joining (NHEJ) efficiency was similar for groups 1 (14/18; 77.7%) and 2 (14/17; 82.3%). In experiment 2, the cleavage (53/76; 69%) and blastocyst rates (30/76; 39%) were significantly higher for the control than for the treated groups (P < 0.01). Among the groups, the lower cleavage and blastocyst rates were for group 4 (20/61; 32.7% and 3/61; 4.9%, respectively) compared with the other electroporation and microinjection groups (P < 0.03). However, NHEJ efficiency was higher for electroporation groups 2 (6/8; 75%), 3 (17/17; 100%), and 4 (2/2; 100%) compared with microinjection (2/15; 13%). In conclusion, electroporation of Cas9/RNP is an efficient alternative to microinjection for gene editing in porcine zygotes.