Optimizing the delivery of CRISPR/Cas9 ribonucleoproteins for efficient genome editing in bovine embryos

Aug 8, 2025Gene

Improving CRISPR gene editing in cow embryos by optimizing protein delivery

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Abstract

CRISPRMAX transfection achieved 30% PRLR-edited blastocysts with an 8% homozygous deletion.

Gene editing efficiency varied with different transfection methods, indicating that method selection is crucial.
Increasing voltage and pulse duration in electroporation improved gene editing but reduced embryo viability.
NEPA21 electroporation with a commercial enhancer achieved up to 47.6% PRLR deletion but resulted in lower embryo cleavage and blastocyst rates.
Combining NEPA21 electroporation with CRISPRMAX lipofection improved editing efficiency to 50%, though with reduced blastocyst rates.
Neon electroporation produced 65.2% PRLR-edited blastocysts, but also led to lower viability metrics.

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To assist in establishing a streamlined and efficient workflow for generating gene-edited bovine embryos, we evaluated three transfection approaches for the delivery of CRISPR Cas9-sgRNA ribonucleoproteins into bovine zygotes: lipofection with Lipofectamine CRISPRMAX, and electroporation using either Neon or NEPA21 electroporation systems. Bos taurus prolactin receptor (PRLR) was used as the target gene. The PRLR editing outcomes were analysed by PCR genotyping and Sanger sequencing of individual embryos at day 8 post-fertilization. CRISPRMAX transfection generated up to 30 % PRLR-edited blastocysts (8 % homozygous deletion), without affecting the embryo cleavage (93 %) and blastocyst rate (39 %) relative to non-transfected controls. For both NEPA21 and Neon electroporation, we found that increasing the voltage, length and number of pulses resulted in enhanced gene editing efficiency but compromised embryo cleavage and blastocyst rates. NEPA21 electroporation with a commercial electroporation enhancer reagent produced up to 47.6 % transfected embryos with the PRLR deletion, but with decreased embryo cleavage (62 %) and blastocyst (18 %) rates. Combining NEPA21 electroporation with CRISPRMAX lipofection enhanced the gene editing efficiency to 50 % (23 % homozygous editing), with 64 % embryo cleavage rate and 18 % blastocyst rate. Notably, Neon electroporation resulted in 65.2 % PRLR-edited blastocysts with 21 % homozygous editing (50% cleavage rate and 10 % blastocyst rate). Additional studies may be necessary to further optimize electroporation parameters to achieve an optimal balance between embryo viability and gene editing efficiency. These outcomes will provide valuable insights for improving gene editing workflows for bovines and may help to promote and accelerate the widespread implementation of genome editing technology in livestock.

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