Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant...

Area of Science:

• Biotechnology
• Genetics
• Reproductive Biology

Background:

• Generating targeted mutant mice is essential for biomedical research.
• The CRISPR/Cas9 system is a key technology for creating these mutants.
• Optimizing gene delivery into sperm is critical for efficient mutant generation.

Purpose of the Study:

• To optimize the CRISPR/Cas9 system's uptake into sperm using methyl β-cyclodextrin-sperm-mediated gene transfer (MBCD-SMGT).
• To investigate the roles of cholesterol and reactive oxygen species (ROS) in sperm's exogenous DNA uptake.
• To establish an efficient method, MBCD-sperm-mediated gene editing (MBCD-SMGE), for generating targeted mutant blastocysts and mice.

Main Methods:

• Sperm cells were incubated with varying concentrations of MBCD and a CRISPR-Cas9 plasmid.
• Functional sperm parameters, extracellular ROS, and plasmid copy numbers per sperm were assessed.
• In vitro fertilization (IVF) was performed, followed by assessment of fertilization, embryonic development, and transfection rates.

Main Results:

• Cholesterol removal via MBCD induced premature acrosomal reactions and increased extracellular ROS.
• MBCD-SMGT resulted in a higher number of transfected motile sperm and increased GFP-positive blastocysts.
• Targeted indels were validated in blastocysts and mice generated using the MBCD-SMGE technique.

Conclusions:

• The MBCD-SMGE technique shows significant potential for efficiently generating targeted mutant mice.
• This method holds promise for advancing disease modeling and improving animal traits.
• Optimized sperm-mediated gene transfer is crucial for efficient genetic engineering in mice.