Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Conditional knockout mouse models are crucial for studying gene function in a tissue-specific manner.
• The creation of floxed alleles, a key component of these models, is often challenging and demands reliable methods.
• Increased demand for floxed mouse models in biomedical research necessitates efficient and economical generation techniques.

Purpose of the Study:

• To provide detailed technical protocols for the reliable and economical creation of floxed alleles.
• To present a comprehensive method for generating floxed alleles using CRISPR-Cas9 technology.
• To establish validated protocols for generating conditional knockout mouse models.

Main Methods:

• Electroporation of single-cell embryos with CRISPR ribonucleoproteins (RNPs) and single-stranded oligodeoxynucleotides (ssODNs).
• Next-generation sequencing (NGS)-based genotyping for allele verification.
• In vitro Cre assay for loxP phasing determination and optional second-round targeting for indel insertion.
• Validation protocols for guide RNAs (gRNAs) and ssODNs before embryo electroporation.

Main Results:

• Successful generation of floxed alleles using the described CRISPR-based method.
• Accurate determination of loxP phasing and indel status in individual blastocysts.
• Demonstrated reliability and predictability in obtaining floxed alleles.
• An alternative strategy for sequential insertion of loxP sites was presented.

Conclusions:

• The presented method offers a reliable, predictable, and timely approach for generating floxed alleles.
• This protocol facilitates the creation of conditional knockout mouse models, advancing biomedical research.
• The validated protocols and strategies aim to overcome current challenges in floxed allele generation.