New software tool provides a way for safer design of genome editing

A team of researchers has developed a software tool called DANGER (Deletious and Anticipatory Guides Assessed by RNA-Sequencing) analysis that provides a means for the safe design of whole-organism genome editing, including the transcriptome. For nearly a decade, researchers have used CRISPR technology for genome editing. However, there are some challenges in using CRISPR. DANGER analysis overcomes these challenges and allows researchers to perform safe on- and off-target assessments without reference genomes. It has potential applications in medicine, agriculture and biological research.

Their work is published in journals Advances in Bioinformatics On August 23, 2023.

Genome editing, or gene editing, refers to technology that allows researchers to alter an organism’s genomic DNA. With this technology, researchers can add, remove, or modify genetic material in the genome.

CRISPR-Cas9 is a well-known gene editing technology. It has a reputation for being more accurate, faster and less expensive than other similar technologies. However, gene editing using CRISPR technology presents some challenges. The first challenge is that phenotypic, or observable, effects caused by unpredictable CRISPR dynamics are not quantitatively observed.

A second challenge is that CRISPR technology typically relies on basic genomic data, including reference genomes. The reference genome is like a template that provides researchers with general information about the genome. Unexpected sequence editing with mismatches may occur. These off-target sites are always unexpected. Researchers therefore need a way to monitor the actual genomic sequence and limit potential off-target effects.

Genome editing design requires a well-characterized genomic sequence. However, genomic information in patients, cancers and non-sentient organisms is often incomplete.”

Kazuki Nakama, Assistant Professor at PTBO Collaborative Research Laboratory, Genome Editing Innovation Center, Hiroshima University

The research team set out to develop a method to deal with the problems of phenotypic effects and dependence on a reference genome. The team’s DANGER analysis software overcomes these challenges The team conducted their risk-versus on- and off-target assessment on RNA-sequencing data using gene-edited samples of human cells and zebrafish brains.

The team demonstrated that the threat analysis pipeline achieves several goals. It identified potential DNA on- and off-target sites in mRNA-transcribed regions in the genome using RNA-sequencing data. It assessed phenotypic effects by deleterious off-target sites based on evidence provided by gene expression changes. It quantified phenotypic risk at the Gene Ontology term level without a reference genome. This breakthrough has shown that DANGER can be analyzed on a variety of organisms, individual human genomes, and atypical genomes caused by diseases and viruses.

The DANGER analysis pipeline identifies genomic on- and off-target sites based on new Transcriptome assembly using RNA-sequencing data. A transcriptome includes a collection of all active gene readouts in a cell. with new Transcriptome assembly, the transcriptome is assembled without the aid of a reference genome. Next, DANGER analysis detects harmful off-targets These are off-target mRNA-transcribed regions that represent decreased expression in edited samples compared to wild-type. Finally, the software quantifies phenotypic risk using gene ontology of off-target lesions. “Our DANGER analysis is a novel software that enables the quantification of phenotypic effects caused by estimated off-targets. Moreover, using our tool new Transcriptome assemblies whose sequences can be constructed from RNA-sequencing data of treated samples without a reference genome, says Hidemasa Bono, a professor at Hiroshima University’s Genome Editing Innovation Center.

Looking ahead, the team hopes to expand their research using DANGER analysis. “We will apply the software to a variety of genome editing samples from patients and crops to clarify phenotypic effects and establish safe techniques for genome editing,” Nakamai said.

Danger Analysis is open source and freely adjustable. The algorithm of this pipeline can therefore be reused for the analysis of different genome editing systems beyond the CRISPR-Cas9 system. It is also possible to increase the specificity of the DANGER analysis by incorporating CRISPR-Cas9-specific off-target scoring algorithms for CRISPR-Cas9. The team believes that the DANGER analysis pipeline will expand the scope of genomic studies and industrial applications using genome editing.

The research team includes Kazuki Nakamai who works at Hiroshima University and PtBio Inc. and Hidemasa Bono who works at Hiroshima University.

This research was funded by the Innovation Center for Bio-Digital Transformation; Open Innovation Platform for Industry-Academia Co-creation (COI-NEXT), Japan Science and Technology Agency COI-NEXT (JPMJPF2010); and the Japan Society for the Promotion of Science Kakenhi (21K17855).