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

MOTIVATION: The CRISPR-Cas9 system has successfully achieved site-specific gene editing in organisms ranging from humans to bacteria. The technology efficiently generates mutants, allowing for phenotypic analysis of the on-target gene. However, some conventional studies did not investigate whether deleterious off-target effects partially affect the phenotype. RESULTS: Herein, we present a novel phenotypic assessment of CRISPR-mediated gene editing: Deleterious and ANticipatable Guides Evaluated by RNA-sequencing (DANGER) analysis. Using RNA-seq data, this bioinformatics pipeline can elucidate genomic on/off-target sites on mRNA-transcribed regions related to expression changes and then quantify phenotypic risk at the gene ontology term level. We demonstrated the risk-averse on/off-target assessment in RNA-seq data from gene-edited samples of human cells and zebrafish brains. Our DANGER analysis successfully detected off-target sites, and it quantitatively evaluated the potential contribution of deleterious off-targets to the transcriptome phenotypes of the edited mutants. Notably, DANGER analysis harnessed de novo transcriptome assembly to perform risk-averse on/off-target assessments without a reference genome. Thus, our resources would help assess genome editing in non-model organisms, individual human genomes, and atypical genomes from diseases and viruses. In conclusion, DANGER analysis facilitates the safer design of genome editing in all organisms with a transcriptome. AVAILABILITY AND IMPLEMENTATION: The Script for the DANGER analysis pipeline is available at https://github.com/KazukiNakamae/DANGER_analysis. In addition, the software provides a tutorial on reproducing the results presented in this article on the Readme page. The Docker image of DANGER_analysis is also available at https://hub.docker.com/repository/docker/kazukinakamae/dangeranalysis/general.