GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases

Area of Science:

• * Molecular Biology
• * Bioinformatics
• * Genomics

Background:

• * CRISPR-Cas9 genome editing offers powerful tools for biological research and genetic disorder therapies.
• * Accurately identifying all DNA sequences targeted by CRISPR-Cas9 nucleases and their cleavage efficiency is crucial for therapeutic safety.
• * Experimental methods like GUIDE-seq enable sensitive, unbiased, genome-wide detection of nuclease cleavage sites.

Purpose of the Study:

• * To develop a flexible bioinformatics software suite for analyzing GUIDE-seq data.
• * To provide an open-source, open-development R package for processing and annotating nuclease cleavage sites.
• * To facilitate the characterization of on-target and off-target cleavage activities for various genome editing nucleases.

Main Methods:

• * Development of the GUIDEseq R package, available via Bioconductor.
• * Implementation of over 60 adjustable parameters for tailored data analysis.
• * Integration of genome annotation for identifying off-target sites near genes.
• * Inclusion of off-target cleavage score prediction and comparison functionalities.

Main Results:

• * The GUIDEseq package offers a flexible platform for analyzing GUIDE-seq data from diverse nuclease applications and species.
• * It allows customization of analysis criteria, including sequence aggregation and peak calling thresholds.
• * The software annotates potential off-target sites overlapping with genes and facilitates comparison between datasets.
• * It outputs read counts and cleavage scores for identified off-target sites, enabling detection of unexpected cleavage activity.

Conclusions:

• * The GUIDEseq package provides a comprehensive solution for analyzing GUIDE-seq data across various nuclease platforms and species.
• * Successful application in analyzing multiple GUIDE-seq datasets demonstrates its utility and robustness.
• * The freely available software, source code, and documentation promote open access and further development.