Format for information and overview on SDN-1 and/or SDN-2 genome edited plants to IBSC. Guidelines for the Safety Assessment of Genome-Edited Plants, 2022” by DBT for SDN-1 and SDN-2 categories of genome-edited plants. This exemption applies to site-directed nuclease (SDN)-1 and SDN-2 categories of genome-edited plants, which are free from exogenously introduced DNA.
The development of genome-modified plants will be carried out under control, until they are freed from the introduced exogenous DNA. Pursuant to the O.M., Department of Biotechnology (DBT), Ministry of Science and Technology announced 'Guidelines for Safety Assessment of Genome Modified Plants, 2022'2 on May 17, 2022 for research and development of genome modified plants in India. The Guidelines provide comprehensive guidance for all categories of GM plants, eg, SDN-1, SDN-2 and SDN-3.
The guidelines provide guidance on regulatory pathways and information/data requirements for the safety assessment of genome-edited plants. The definition of SDN-1 and SDN-2 genome-edited plants is provided in the box below. To facilitate regulatory review for research and development of genome-edited plants falling under categories SDN-1 and/or SDN-2 until they are free from exogenously introduced DNA.
The generation in which the genome-edited lines were found to be free of exogenously introduced DNA.
Import of SDN-1 and SDN-2 genome edited plants/seeds/ propagules for research, testing, and product development
On receipt of RCGM acceptance of IBSC proceedings, a letter will be issued to the applicant (importer) by IBSC confirming that the subject material is exempt from regulation under the Rules, 1989 in the format given in Annexure- V. Acceptance of IBSC meeting minutes by RCGM must be attached to the application for plant quarantine import permit.
Record keeping
Flow chart for seeking exemption for SDN-1 and SDN-2 genome edited plants
Protocols to show that the genome edited plants are free from exogenous introduced DNA
Absence of selection/scorable marker
Overlapping PCR/ nested PCR
Genomic DNA of the parental genotype spiked with no more than 1/1000 (w/w) of the purified full-length vector DNA used to develop the modified genome line (using the same set of primers as used for overlap PCR). The amount of template DNA and PCR conditions should be such that a clear band of the expected size is visible in all positive controls. No amplification should be detected in any of the reactions with primers directed against the exogenous inserted DNA for the final modified genome line, while clear amplification should be detected in all positive controls by stained agarose gel electrophoresis with ethidium bromide.
Use of other methods
Nomenclature for the genome edited lines that are free from exogenous introduced DNA
Review of the SOPs
Annexure-II
Details about the investigator(s) and the institution Name of the Principal Investigator
Details about the genome edited plant(s) S
Data on the phenotypic expression of the target trait(s), as assessed under containment conditions, if applicable. By sequencing the parental and modified allele(s) using Sanger or other Sequencing technologies with a minimum 10X coverage of the edited region(s) and - base quality of minimum Phred score 30 11b. Whether self-pollination and/or backcrossing has been performed to separate the exogenously introduced DNA.
Provide evidence to confirm the absence of exogenously introduced DNA in genome-edited plants by phenotypic selection (sensitivity to herbicide/antibiotic, or absence of scorable marker) and use of overlapping PCR/Nested PCR/other appropriate methodology (as described in Chapter 5). In case, unintended phenotypic changes were observed on the genome-edited plant regardless of whether it was selected/separated. In the case of nutritional traits: provide data on the intended nutritional trait compared to the parent line.
Annexure-III
In the case of nutritionally related traits, whether data is provided on the targeted nutritional trait compared to the parental line.
Annexure-IV Format for seeking permission to import SDN-1 and/or SDN-2 genome
Details about the Importer Applicant information
Annexure-IV Format for requesting permission to import the SDN-1 and/or SDN-2 genome. molecular tools) including maps and nucleotide sequences. Data on the phenotypic expression of the trait(s) of interest, as assessed under containment conditions, if applicable 11. By sequencing the parental and modified allele(s) using Sanger or other sequencing technologies with a coverage of at least 10x of the edited region(s) ) and base quality of a minimum Phred score 30.
Whether a DNA-free method such as the RNA-protein complex was used for genome editing. Provide evidence to confirm the absence of exogenously introduced DNA in genome-edited plants by phenotypic selection (sensitivity to herbicide/antibiotic, or absence of scorable marker) and use of overlapping PCR/Nested PCR/other appropriate methodology (as described in Chapter 5).
Annexure-V Format for communicating confirmation of the absence of exogenous introduced
GLOSSARY
Sources: The definitions of key terms, as in the glossary, are taken from national rules and guidelines, as well as from documents of international bodies such as the Food and Agriculture Organization (FAO). Off-target mutations Mutations in the genome that are caused by programmable nuclease(s) at sites other than the target site in the genome. Phenotype The visible and/or measurable characteristics of an individual (related to one or more traits) that reflect the response of a particular genotype in a particular environment.
Prime editing A genome editing method for targeted changes in the DNA sequence such as small insertions, deletions and base swaps in a precise manner using RNA repair template without requiring the creation and repair of DSBs. In prime editing, an RNA template is used to guide the repair of the target locus through the activity of a reverse transcriptase. Segregation For genes, the separation of pairs of alleles from each other and their resulting assortment in different cells by meiosis.
For chromosomes, the separation and re-assortment of the two homologs in anaphase of the first meiotic division. Site Directed Nuclease (SDN) Engineered DNA nucleases that are programmed to specific locations in the genome of an organism where they break a DNA chain by separating nucleotides. Unintended effect An effect considered to be a consistent difference between the genome-edited plant and its parent line that goes beyond the primary intended effect(s) of the genome editing.
Addendum
Applicability of information and data requirements, as listed in the
Guidelines for the safety assessment of genome edited plants, 2022” by DBT for SDN-1 and SDN-2 categories of genome edited plants
27. loci and their direct or indirect effects. (also called off-target mutations) generated in the genome editing process would be very few compared to those from conventional mutagenesis procedures. If any of the off-target mutations were to affect the plant phenotype, the developer would segregate and remove phenotypically foreign plants that might arise during the genome editing process. Further, the mutant lines developed will also be evaluated in field trials and if any undesirable trait is identified, they will be discarded at that stage.
This information is considered sufficient and would be consistent with the procedures carried out during the variety development process and mutagenesis procedures. Therefore, molecular expression profiling of the target gene is considered unnecessary. Backcross for a sufficient number of generations to remove any off-target changes in the case of genome-edited plants.
In the case of perennial plants or plants that reproduce mainly through vegetative propagation, additional molecular data may be needed on a case-by-case basis. In the case of off-target changes, information showing that they are not different from any variant for the off-target trait found in the species or among segregating progeny in conventional breeding materials. The number of off-target mutations generated in the genome editing process would be very few compared to conventional mutagenesis procedures.
If any of the off-target mutations were to affect the plant phenotype, a developer would segregate and remove phenotypically foreign plants that might arise during the genome editing process. When the intended change is The SOPs indicate that in the case of nutritional. The SDN-1 and/or SDN-2 genome-modified plants free from exogenously introduced DNA would be treated in the same way as in the case of a product of conventional mutation breeding.
ACKNOWLEDGEMENTS
END OF THE DOCUMENT
