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Viability-Based Assessment Reveals True Efficiency of CRISPR-Cas9 Transfection Methods in Rabbit Spermatozoa.

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  • 1Commonwealth Scientific and Industrial Research Organisation (CSIRO), Health & Biosecurity, Acton, Australia.

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Summary

Lipofection improves CRISPR-Cas9 delivery into viable sperm, overcoming limitations of electroporation for efficient genome editing. This method enhances sperm transfection for potential applications in rabbit genome engineering.

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Area of Science:

  • Mammalian genome engineering
  • Reproductive biology
  • Gene editing technologies

Background:

  • CRISPR-Cas9 enables mammalian genome engineering but faces challenges with *in vivo* efficiency and mosaicism.
  • Sperm-mediated delivery is a potential strategy to improve genome editing efficiency and reduce mosaicism.

Purpose of the Study:

  • To compare electroporation and lipofection for delivering CRISPR-Cas9 ribonucleoproteins into viable rabbit spermatozoa.
  • To assess the impact of these methods on sperm viability and motility.

Main Methods:

  • Utilized Fluorescence-Activated Cell Sorting (FACS) to evaluate transfection efficiency.
  • Compared Neon NxT electroporation and Lipofectamine CRISPRMAX lipofection for introducing CRISPR-Cas9 ribonucleoproteins into rabbit spermatozoa.

Main Results:

  • Electroporation showed poor efficiency when only viable spermatozoa were considered.
  • Lipofectamine CRISPRMAX significantly improved transfection efficiency with minimal impact on sperm viability and motility.
  • Distinguishing true transfection of viable sperm from total transfection is crucial.

Conclusions:

  • Lipofection is a more effective method than electroporation for sperm-based genome editing reagent delivery.
  • This finding highlights lipofection as a promising alternative for sperm-mediated genome engineering, particularly in rabbits.