New publication: Gene-editing platform for characterizing target mutations in plants


Researchers from GenØk–Centre for Biosafety and colleagues from the Federal University of Santa Catarina (UFSC) in Brazil, developed a fast and efficient model system to identify a range of possible target mutations in plant species. The platform consists of three major steps, the isolation of plant cells, delivery of CRISPR-Cas9 as proteins and further sequencing analysis. All methods applied are non-mutagenic with the exception of CRISPR-Cas9, which enables the identification of side-effects derived from this technique alone.

This efficient and relatively easy assay method will be useful for characterizing different types of mutations generated by this CRISPR-Cas9 genome-editing technique in virtually any plant species.

The cell-based model provides new risk hypothesis for gene-edited plants
The commercialization of gene-edited plants is regulated in Europe through the ‘Genetically Modified Organisms’ Directive 2001/18/EC which requires pre-market full risk assessment including detection and identification methods. Therefore, validated and harmonized detection methods are crucial for the enforcement of the law.

Our platform is designed to test the genetic modification outcome in thousands of plant cells that have been exposed to CRISPR-Cas9 editing tool without a DNA repair template.  CRISPR-Cas9 editing tool is capable of producing several double-stranded DNA breaks in living cells that will trigger the cell natural repair mechanism which will then result in a genetic modification, also called mutations. Because this is an unsupervised approach, the outcome is unknown. The analysis of a large amount of cell data shows the range of potential changes that could be present in an organism that has been submitted to similar editing techniques. Therefore, new risk hypotheses can be drawn from data using the same guide RNA sequences to test the range of mutations that are present. In other words, it will provide new risk hypotheses based on the potential of this genetic engineering technique instead of limiting the hypothesis to the intended modification in the final product.

The next step is to apply the same platform to investigate off-target effects
The platform has so far been tested at target sequence regions. But the next step is to test other parts of the host genome that could be affected by the CRISPR-Cas9 technique. Therefore, researchers will adapt the sequencing strategy to cover the entire nuclear genome. Several analytical limitations are foreseen, and these will be accounted through an uncertainty analysis and further reported.

The platform is a first step is designing gene-editing experiments that are fully dedicated to study adverse effects of CRISPR-Cas9 systems in plants and how to use this knowledge to test the safety of edited organisms to be commercialized in the future.

The new study:
Sant’Ana RRA, Caprestano CA, Nodari RO, Agapito-Tenfen SZ. PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts. Genes (Basel). 2020;11(9):E1029. Published 2020 Sep 2. doi:10.3390/genes11091029.