Optimizing CRISPR precision in mouse embryos via microhomology-mediated end joining-dominant targeting

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Developmental Biology

Background:

• CRISPR/Cas9 gene editing in mice frequently results in unpredictable, mosaic mutations due to reliance on non-homologous end joining (NHEJ).
• Existing methods lack predictability in generating precise genetic modifications in founder (F0) animals.

Purpose of the Study:

• To develop an enhanced genome editing strategy for mice that improves the predictability and uniformity of gene edits.
• To reduce mosaic mutations in founder animals generated via CRISPR/Cas9 technology.

Main Methods:

• A hybrid approach combining in silico prediction software with in vitro validation in mouse embryonic stem cells (mESCs).
• Pre-selection of guide RNAs (gRNAs) predicted to favor microhomology-mediated end joining (MMEJ) repair pathways.
• Validation of selected gRNAs in mESCs to predict in vivo editing outcomes before embryo injection.

Main Results:

• In vitro validation in mESCs accurately predicted in vivo mutation patterns observed in later embryonic stages.
• The integrated pipeline successfully generated founder (F0) mice with highly uniform genotypes for the Tyr and Fgf10 genes.
• The MMEJ-dominant strategy significantly enhanced genotype uniformity compared to standard CRISPR/Cas9 approaches.

Conclusions:

• The developed hybrid genome editing strategy enhances the predictability and reproducibility of CRISPR-based gene editing in mice.
• This approach offers a more reliable method for generating genetically uniform founder animals.
• The strategy has the potential to reduce the number of animals required for gene editing studies.