Objective:
This work aims to determine how genomic population structure and environmentally driven developmental variability can inform a reproducible molecular lineage framework for Cannabis sativa, and how such baselines support the evaluation of targeted genome-editing approaches.

Methods:
Commercial and legacy accessions were analyzed using whole-genome resequencing (30–40× coverage), high-density SNP genotyping, and population-structure analyses. Genetic clusters were compared with common strain names and indica/sativa labels.

Genome-editing experiments used two nuclease platforms—CRISPR/Cas9 and an alternative non-Cas9 system—delivered as ribonucleoprotein complexes into de novo and callus-based regeneration pipelines. To assess editing efficiency and developmental stability, two reporter loci were targeted: a GFP insertion cassette and a loss-of-function mutation in the PDS (phytoene desaturase) gene, a standard albino reporter in plant editing. Edits were confirmed by deep amplicon sequencing and fluorescence or phenotype imaging.

Results:
Genomic analyses revealed substantial naming redundancy and polyphyly across commercial labels, although some shallow but recurrent structure aligned with certain widely used categories. Environmental treatments highlighted strong developmental plasticity, underscoring the need for genomic baselines when interpreting phenotypic outcomes.

Both nuclease systems produced consistent reporter edits. GFP-positive tissues displayed stable fluorescence across regeneration stages, and PDS knockouts generated clear albino phenotypes, indicating effective biallelic disruption. Editing frequencies and developmental behavior were similar across the two nuclease platforms, suggesting that the main limitations originate from the regeneration biology of cannabis rather than from the editors themselves.

Establishing reproducible genomic lineages provides the necessary framework for accurately interpreting edited phenotypes, allowing true editing effects to be distinguished from background genetic or environmental variability.

Conclusions
1. Genomic lineage frameworks improve biological accuracy without displacing the cultural and communicative value of traditional cultivar names.
2. Interpreting edited phenotypes requires standardized genomic references due to high developmental and environmental influences.
3. Genome editing in cannabis performs similarly across Cas9 and non-Cas9 nucleases, but predictable outcomes depend on validated genetic baselines and optimized regeneration systems.

Taken together, these results outline an integrated foundation for authentication, breeding, and precision genome engineering in Cannabis sativa.

Learning Objectives: 

• Understand how standardized genomic baselines enable accurate interpretation of edited phenotypes, and evaluate the advantages of gene editing over conventional breeding using current plant biotechnology platforms