Amount of Cas9 protein introduced into mouse embryos via electroporation affects the genome-editing rate

• Main Authors: Yui WAKE (Author), Christopher A. VAKULSKAS (Author), Steve E. GLENN (Author), Takehito KANEKO (Author)
• Format: Book
• Published: The Society for Reproduction and Development, 2022-07-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 0916-8818; 1348-4400; 10.1262/jrd.2022-067
• Keywords: crispr/cas; electroporation; embryos; genome editing; mouse; Reproduction; QH471-489; Internal medicine; RC31-1245
• Source: The Journal of Reproduction and Development, Vol 68, Iss 5, Pp 307-311 (2022)

Genetically engineered animals can be produced quickly using genome editing technology. A new electroporation technique, technique for animal knockout system by electroporation (TAKE), aids in the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of microinjection. It is difficult to confirm nuclease delivery into embryos after electroporation using the conventional TAKE method. We previously reported the successful visualization of fluorescently-labeled tracrRNA in embryos after electroporation Cas9 paired with the crRNA:tracrRNA-ATTO550 duplex. However, the amount of fluorescence signal from labeled tracrRNA in embryos did not correlate with the genome editing rate of the offspring. This study examined the visualization of Cas9 protein in embryos after electroporation and its correlation with the genome editing rate of the offspring using a fluorescent Cas9 fusion protein. The fluorescent Cas9 protein was observed in all embryos that survived following electroporation. We found that the efficiency of Cas9 protein delivery into embryos via electroporation depended on the pulse length. Furthermore, we demonstrated that the amount of fluorescent Cas9 protein detected in the embryos correlated with the genome editing efficiency of the embryos. These data indicate that the TAKE method using fluorescently-labeled nucleases can be used to optimize the delivery conditions and verify nuclease delivery into individual embryos prior to embryo transfer for the efficient production of genome-edited animals.