Crisflash: open-source software to generate CRISPR guide RNAs against genomes annotated with individual variation.

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• CRISPR/Cas9 gene editing relies on short guide RNAs (sgRNAs) for precise DNA targeting.
• Existing sgRNA design tools often lack flexibility, primarily supporting standard reference genomes and small-scale applications.

Purpose of the Study:

• To develop a high-performance software tool, Crisflash, for efficient sgRNA design and off-target analysis.
• To overcome the limitations of current tools by enabling rapid design against any genome and incorporating variant data.

Main Methods:

• Crisflash was developed as a flexible software tool for sgRNA design and off-target discovery.
• The tool was optimized for speed and performance, capable of analyzing any sequenced genome or genome sequences.
• Incorporation of user-supplied variant data allows for enhanced guide accuracy.

Main Results:

• Crisflash demonstrates over an order of magnitude improvement in speed compared to existing tools, even on a single CPU core.
• The software efficiently and robustly scores potential off-target effects for all candidate sgRNAs.
• Enables rapid design of CRISPR guides against diverse genomic targets.

Conclusions:

• Crisflash provides a significant advancement in CRISPR/Cas9 sgRNA design, offering speed, flexibility, and accuracy.
• The tool is well-suited for both small-scale and large-scale genomic research, including projects with non-standard reference genomes or specific variant data.