ABSTRACT
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. 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 (GO) 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.
Competing Interest Statement
K.N. was employed by PtBio Inc. H.B. was a consultant for PtBio Inc. H.B. has a financial interest in PtBio Inc.
Footnotes
Kazuki Nakamae, Ph.D. Genome Editing Innovation Center, Hiroshima University, 3-10-23 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan E-mail: kazuki-nakamae{at}hiroshima-u.ac.jp Tel: +81-82-424-4013 Fax: +81-82-424- 3990
Hidemasa Bono, Ph.D. Graduate School of Integrated Sciences for Life, Hiroshima University, 3-10-23 Kagamiyama, Higashi-Hiroshima city, Hiroshima 739-0046, Japan E-mail: bonohu@hiroshima-u.ac.jp Tel.: +81-82-424-4013
A few descriptions were added to Material & Methods and Result & Discussion