CRISPR-Cas9 Induced Knockout of BEL5 in Tetraploid Potato: Optimized Methodology via Repeated de novo Regeneration and Impact on Tuberization

Technical Summary: CRISPR-Cas9 Induced Knockout of BEL5 in Tetraploid Potato

Problem Statement
The application of CRISPR-Cas9 genome editing in cultivated potato (Solanum tuberosum ssp. tuberosum) faces significant hurdles due to its tetraploid, highly heterozygous genome and vegetative propagation. Achieving a complete knockout of all four alleles of a target gene is difficult because standard transformation often results in mosaicism (where only a subset of cells or alleles are edited) or incomplete editing across alleles. Furthermore, the lack of haplotype-resolved whole-genome data for many commercial cultivars complicates the design of guide RNAs (gRNAs) that target all alleles while minimizing off-target effects. Additionally, reliable detection of mutations in polyploids is challenging, particularly when phenotypic changes are subtle or absent, necessitating robust molecular validation strategies.

Methodology
The authors developed an optimized workflow to achieve complete BEL5 gene knockout in the tetraploid cultivar 'Désirée' (and attempted it in 'Otava'). The methodology involved several key steps:

1. Genomic Characterization and gRNA Design: For 'Désirée', where a fully assembled genome was unavailable, the authors reconstructed four BEL5 alleles by mapping unassembled Illumina short-read data to the reference BEL5 sequence. They validated these reconstructions using long-read Oxford Nanopore sequencing. Two gRNAs were designed to target the 1st and 4th exons, ensuring coverage of all four alleles. Restriction sites within the target sequences were leveraged for initial screening.
2. Transformation and Regeneration: The study utilized Agrobacterium-mediated transformation of leaf explants followed by de novo organogenesis. To overcome the limitations of T0 generation mosaicism and incomplete editing, the authors employed a strategy of repeated de novo regeneration. Leaf explants from selected T0 plants were subjected to a second round of tissue culture. This approach aimed to exploit the dynamic chromatin remodeling and epigenetic reprogramming associated with dedifferentiation to increase target site accessibility and induce new mutations in previously unedited alleles.
3. Mutation Detection and Validation: A multi-tiered screening approach was used:
   • CAPS Analysis: Cleaved Amplified Polymorphic Sequences analysis was used for rapid initial screening of PCR products to detect disruptions in restriction sites.
   • Sanger Sequencing & TIDE: Amplicons were sequenced and analyzed using Tracking of Indels by DEcomposition (TIDE) software to estimate editing efficiency and identify frameshifts.
   • Nanopore Amplicon Sequencing: For final validation, the entire BEL5 gene and flanking UTRs were sequenced using Oxford Nanopore technology. This high-depth sequencing allowed for allele-resolved analysis, detection of large insertions/deletions, and identification of minor unedited alleles that might escape detection by other methods.
4. Phenotypic Analysis: Complete knockout lines were grown in hydroponic systems under short-day (inductive) conditions to monitor tuberization onset, and in soil under long-day conditions to assess total yield.

Key Results

• Editing Efficiency: The initial T0 generation showed mixed editing, with most lines containing a combination of edited and unedited alleles. However, the strategy of repeated de novo regeneration significantly improved outcomes. In the T1 generation (descendants of T0 line 60), the authors identified five lines (60-4, 60-5, 60-25, 60-28, 60-37) that appeared to be fully edited.
• Novel Mutations: The second round of regeneration promoted the emergence of de novo mutations in alleles that were previously unedited in the T0 progenitor. One line (60-37) was found to retain a very small proportion of unedited sequence at one allele, highlighting the necessity of deep sequencing for confirmation.
• Mosaicism: Analysis of six consecutive leaves in the regenerated lines indicated negligible sectorial mosaicism, suggesting that the regeneration process effectively homogenized the genetic background.
• Phenotypic Impact: BEL5 knockout plants exhibited a delayed onset of tuberization under short-day conditions compared to wild-type plants. However, by the end of the growing period, the mutant plants produced a total tuber yield comparable to wild-type plants. The knockout did not prevent tuberization entirely, nor did it significantly alter whole-plant biomass.
• Genotype Specificity: The study noted that the 'Otava' cultivar was difficult to regenerate via this protocol, yielding only one transgenic line with no detectable edits, underscoring the genotype-dependent nature of transformation efficiency.

Significance and Claims
The paper claims to provide a methodological framework for achieving complete CRISPR-Cas9-mediated gene knockouts in vegetatively propagated polyploid crops, specifically addressing the challenges of heterozygosity and mosaicism. The authors propose that repeated de novo regeneration is a critical strategy for promoting the emergence of new mutations in unedited alleles, thereby facilitating the generation of fully edited lines without the need for sexual segregation.

Functionally, the study offers new insight into the role of BEL5 in modern cultivated potato. While previous studies in other genotypes suggested BEL5 is a key positive regulator of tuber initiation, this study demonstrates that in the tetraploid cultivar 'Désirée', BEL5 is dispensable for tuber development but plays a regulatory role in the timing of tuber onset. The results suggest that the tuberization network in cultivated potato is robust and capable of compensating for the loss of BEL5, likely through redundant pathways involving other signals such as SP6A.

The authors emphasize that their work highlights the importance of using high-resolution sequencing (such as Nanopore) to validate complete knockouts in polyploids, as standard Sanger sequencing or CAPS analysis may miss low-frequency unedited alleles. They conclude that while BEL5 influences the timing of tuberization, its absence does not preclude successful tuber formation in modern potato varieties.