A team of researchers has developed a software tool called DANGER (Deleterious and ANticipatable Guides Evaluated by RNA-sequencing) analysis that provides a way for the safer design of genome editing in all organisms with a transcriptome. The DANGER analysis overcomes these challenges and allows researchers to perform safer on- and off-target assessments without a reference genome. CRISPR-Cas9 is a well-known gene editing technology. However, gene editing using CRISPR technology presents some challenges. The first challenge is that the phenotypic, or observable, effects caused by unexpected CRISPR dynamics are not quantitatively monitored. A second challenge is that the CRISPR technology generally depends on basic genomic data, including the reference genome.
So researchers need a way to observe factual genomic sequences and limit potential off-target effects. The team demonstrated that the DANGER analysis pipeline achieves several goals. It detected potential DNA on- and off-target sites in the mRNA-transcribed region on the genome using RNA-sequencing data. It evaluated phenotypic effects by deleterious off-target sites based on the evidence provided by gene expression changes. It quantified the phenotypic risk at the gene ontology term level, without a reference genome. The DANGER analysis pipeline identifies the genomic on- and off-target sites based on de novo transcriptome assembly using RNA-sequencing data. A transcriptome includes a collection of all the active gene readouts in a cell. With de novo transcriptome assembly, the transcriptome is assembled without the help of a reference genome.
Next, the DANGER analysis identifies the deleterious off-targets. These are off-targets on the mRNA-transcribed regions that represent the downregulation of expression in edited samples compared to wild-type ones. Finally, the software quantifies the phenotypic risk using the gene ontology of the deleterious off-targets. Researchers said they will apply the software to various genome editing samples from patients and crops to clarify the phenotypic effect and establish safer strategies for genome editing. DANGER analysis is open-source and freely adjustable. So the algorithm of this pipeline could be repurposed for the analysis of various genome editing systems beyond the CRISPR-Cas9 system. It is also possible to enhance the specificity of DANGER analysis for CRISPR-Cas9 by incorporating CRISPR-Cas9-specific off-target scoring algorithms.
