Available online 2 April 2025, 117415

CRISPR CAS9 ribonucleoprotein-electroporation is a feasible strategy to knock out COPA

Functional COPA is necessary for development to the 8-cell stage in bovine embryos

Functional COPA is necessary for development to the blastocyst stage in bovine embryos

CRISPR CAS9 ribonucleoprotein-electroporation in the presence of a repair template is a feasible strategy to realise knock in events into the COPA gene sequence

Functional COPA after homozygous knock in of silent mutations rescues developmental capacity of the embryo

Embryonic genome activation is divided into a minor and a major wave of transition to endogenous transcription. In bovines, minor genome activation begins early in the 2-cell stage and is completed by the 8-cell stage when major genome activation becomes dominant. While the activation of genes known to regulate early development have been studied extensively, genes involved in more central cellular functions have not been examined. Taking advantage of the CRISPR Cas9 system, the present study investigated the effect of knocking out the Golgi retrograde protein transporter COPA on early bovine development. After the electroporation of presumptive zygotes with Cas9 ribonucleoproteins targeting COPA exon 6, sequences of 2 (11%) and 4-cell (16%) embryos showed knockouts of COPA whereas 8-cell embryos and blastocysts did not, demonstrating that COPA is necessary for development to the 8-cell stage and beyond. Using a repair template containing silent mutations along the target site, COPA loss of wildtype was observed in 5 blastocysts, with successful knock-in of the template on at least one allele. This shows that an edited yet functional copy of COPA can save the developmental capacity of the embryo and demonstrates that Cas9 activity at the target region itself is not responsible for the loss of function. Together, the present study revealed that COPA is necessary for embryonic development, and that the timing of this necessity is before major genome activation onset. More generally, this study further demonstrates the utility of genome editing within reproductive biotechnology for the interrogation of gene function and early embryonic development.

CRISPR

IVF

bovine

electroporation

COPA

EGA

embryo

© 2025 The Authors. Published by Elsevier Inc.