Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• CRISPR genome editing facilitates genomic locus evaluation using single guide RNAs (sgRNAs).
• Existing computational tools for sgRNA design and data analysis are often isolated and difficult to integrate into laboratory workflows.
• A need exists for a unified protocol bridging computational and experimental aspects of CRISPR experiments.

Purpose of the Study:

• To present a detailed protocol for both computational and benchtop implementation of arrayed and/or pooled CRISPR genome-editing experiments.
• To provide a user-friendly method for sgRNA design, experimental execution, and high-throughput sequencing data analysis.
• To make CRISPR genome editing accessible to non-experts and facilitate data analysis for diverse biological backgrounds.

Main Methods:

• Utilized CRISPOR for designing and evaluating single guide RNA (sgRNA) sequences.
• Implemented a detailed benchtop protocol for conducting arrayed and pooled CRISPR genome-editing experiments.
• Employed CRISPResso for the analysis of deep-sequencing data to assess genome-editing outcomes.

Main Results:

• The protocol enables the design and execution of arrayed and pooled CRISPR experiments within 4-5 weeks, even for non-experts.
• Computational data analysis using CRISPResso can be completed in 1-2 days via web-based or command-line versions.
• Successfully integrated computational sgRNA design with experimental validation and sequencing data analysis.

Conclusions:

• This protocol streamlines the entire CRISPR genome-editing workflow, from design to data analysis.
• It enhances the accessibility and efficiency of CRISPR-based genomic evaluations for a broader range of researchers.
• The integrated approach empowers non-computational biologists to perform and analyze complex CRISPR experiments effectively.