Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics Engineering

Background:

• CRISPR/Cas9 is crucial for generating rodent models.
• Pronuclear microinjection is the standard method for delivering editing reagents into zygotes.
• Limitations of pronuclear injection include potential embryo damage and clogging.

Purpose of the Study:

• To describe an alternative method for delivering CRISPR/Cas9 reagents into zygotes.
• To present piezo-driven cytoplasmic microinjection as a superior technique.
• To highlight improvements in embryo survival and editing efficiency.

Main Methods:

• Detailed description of the piezo-driven cytoplasmic microinjection technique.
• Comparison with standard pronuclear microinjection.
• Use of Cytochalasin B to soften zygotes.

Main Results:

• Piezo-driven microinjection demonstrates superior embryo survival and birth rates.
• Wider needles allow larger reagent volumes, increasing targeting efficiency.
• Elimination of clogging issues associated with pronuclear injection.
• Potential enhancement of knockin efficiency by Cytochalasin B.

Conclusions:

• Piezo-driven cytoplasmic microinjection is a more effective method for generating genetically modified rodent models.
• This technique offers significant advantages over pronuclear microinjection for CRISPR/Cas9 applications.
• The method holds promise for improving the generation of research models.