CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 is a powerful tool for genome editing, widely used for creating loss-of-function and knock-in mutations.
• Current CRISPR/Cas9 tools often require sequential, single-gene analysis, limiting scalability for large research programs.

Purpose of the Study:

• To develop a scalable CRISPR/Cas9 platform (CRISPRtools) that facilitates gene ablation through exon deletions.
• To streamline the design and implementation of CRISPR/Cas9 for large-scale genetic studies.

Main Methods:

• Utilizing paired guide RNAs to flank and delete critical exons or genomic regions.
• Ranking guide pairs based on efficiency scores and off-target analyses.
• Implementing microinjection and electroporation in mouse zygotes for proof-of-principle experiments.

Main Results:

• Demonstrated the effectiveness of the paired guide RNA strategy for generating precise exon deletions.
• Successfully deleted critical exons in mouse zygotes using microinjection and electroporation.
• Provided a concise format for rank-ordered guide pairs for efficient downstream use.

Conclusions:

• The CRISPRtools platform and exon deletion strategy significantly enhance the scalability and efficiency of CRISPR/Cas9-mediated gene knockout.
• This approach simplifies the characterization of genetically modified animals, particularly for mouse models.
• Facilitates large-scale genomic research programs by optimizing CRISPR/Cas9 experimental design and implementation.