DANGER analysis: risk-averse on/off-target assessment for CRISPR editing without a reference genome

Area of Science:

• Genomics and Bioinformatics
• Molecular Biology
• Gene Editing Technologies

Background:

• CRISPR-Cas9 enables precise gene editing but may cause unintended off-target mutations affecting phenotypes.
• Conventional studies often overlook the impact of deleterious off-target effects on observed gene editing outcomes.
• A robust method is needed to assess both on-target and off-target effects for accurate phenotypic analysis.

Purpose of the Study:

• To introduce a novel bioinformatics pipeline, Deleterious and ANticipatable Guides Evaluated by RNA-sequencing (DANGER) analysis.
• To enable quantitative assessment of phenotypic risks associated with CRISPR-mediated gene editing, including off-target effects.
• To facilitate safer genome editing design across diverse organisms.

Main Methods:

• Utilized RNA-sequencing (RNA-seq) data to identify genomic on/off-target sites impacting mRNA expression.
• Developed a bioinformatics pipeline to quantify phenotypic risk at the gene ontology (GO) term level.
• Employed de novo transcriptome assembly for genome-editing assessments without requiring a reference genome.

Main Results:

• DANGER analysis successfully detected off-target sites in RNA-seq data from gene-edited human cells and zebrafish brains.
• The pipeline quantitatively evaluated the contribution of deleterious off-targets to transcriptome phenotypes in edited mutants.
• Demonstrated risk-averse on/off-target assessment capabilities, even in the absence of a reference genome.

Conclusions:

• DANGER analysis provides a robust method for assessing the safety and potential phenotypic consequences of CRISPR gene editing.
• The pipeline is applicable to a wide range of organisms, including non-model species, human genomes, and viral/disease genomes.
• Facilitates the design of safer and more reliable genome editing strategies for research and therapeutic applications.