Molecular and genetic control of gene-edited plants: methodological approaches

BACKGROUND: Breakthroughs in genome editing technology have made it possible to precisely alter target nucleotides in plant DNA/RNA. Such changes can be identical or comparable to natural mutations, or achieved through conventional mutagenesis, providing the scientific basis for recognizing gene-edited (GE) plants of this category as analogous to plants created through traditional breeding. Risk assessment of off-target editing effects, as well as the methodology and criteria for distinguishing GE plants from transgenic ones, remain unresolved issues for the scientifically based regulation of plant genetic engineering in Russia.

AIM: This study aimed to develop methodological approaches and a decision-making tree for determining the status of plants obtained using genome editing technology, allowing for the registration of the GE plant and its subsequent cultivation.

METHODS: The objects of this analytical study are GE organisms of plant origin.

RESULTS: The result of this work is a comprehensive strategy for minimizing the risks of off-target editing effects and assessing the biosafety of GE plant, namely: a) “safe design” at the concept stage; b) molecular genetic analysis of the obtained GE plants using instrumental methods; c) analysis of the data required for the biosafety assessment of SDN-1 and SDN-2 types of GE plants; d) criteria and a decision-making tree for determining the status of plants obtained using genome editing technology. For instrumental confirmation a modified plant’s status, we proposed to analyze by PCR/RT-PCR of the regions around target editing sites with a length of 1000 base pairs, centered relative to the break site, with subsequent sequencing, and for a more in-depth analysis, the k-mer method.

CONCLUSION: The developed methodological approaches, criteria, requirements, and decision tree will enable the classification of plants obtained using genome editing technology. Thus, the ability to determine whether a plant is transgenic (SDN-3) or gene-edited (SDN-1, SDN-2) will enable the establishment of appropriate regulatory and control measures.