Injury of Genome-Edited Mouse Embryos by Electroporation

Abstract

Background: CRISPR-Cas9-based mouse gene modification, with its simplicity and higher recombination rate compared to embryonic stem cell methods, often involves electroporation into fertilized eggs; however, concerns about its impact on embryonic development. Methods: We conducted embryo culture experiments to assess the developmental impact of electroporation. To determine whether electroporated genome-edited embryos could actually implant in the uterus and become pups, embryo transfer experiments through the oviduct was also conducted. Viability and knock-in efficiency in resulting offspring were analyzed. Results and Conclusion: Electroporated embryos exhibited a significantly lower development rate (median 48.33%, interquartile range 38.03–59.66%) compared to non-electroporated embryos (median 85.00%, interquartile range 69.44–97.50%) at 96 hours post-electroporation. This decrease was consistent in electroporated embryos without the CRISPR-Cas9 complex (median 53.33%, interquartile range 33.44–69.08%, at 96 hours post-electroporation). Despite reduced viability, knock-in efficiency remained high at 62.82%. It is possible to achieve high recombination rates by combining electroporation and genome editing, but increasing the number of embryos to be transferred is required. These findings will be helpful in applications such as gene therapy and livestock genome editing.journal articl