# CRISPR/Cas9-Mediated pds Knockout in Potato Reveals Network-Level Transcriptomic Reorganization Beyond Pigment Loss

## TL;DR

Editing the pds gene in potato with CRISPR/Cas9 causes widespread changes in gene activity beyond just loss of color, revealing complex metabolic and regulatory shifts.

## Contribution

The study reveals that pds knockout in potato leads to broad transcriptomic changes beyond pigment biosynthesis, challenging its use as a simple visual editing marker.

## Key findings

- Albino potato tissues had over 9700 differentially expressed genes compared to wild-type and non-albino edited tissues.
- Transcriptomic changes included suppression of photosynthesis and activation of stress responses and secondary metabolism.
- Weighted gene co-expression network analysis identified distinct regulatory modules linked to albinism and transgenic background effects.

## Abstract

Background: The phytoene desaturase gene is a classical visual marker for validating CRISPR/Cas9 genome editing in plants, as its loss of function produces a readily scorable albino phenotype. While the biochemical basis of pigment loss is well established, it remains unclear whether pds knockout elicits transcriptomic changes extending beyond carotenoid biosynthesis. Resolving this question is essential for correctly interpreting pds-based editing outcomes and for assessing the robustness of phenotype-only screening approaches. Methods: A CRISPR/Cas9 editing platform targeting pds was established in diploid potato. Albino, non-albino edited, and wild-type tissues were subjected to RNA-seq profiling. Differential expression, functional enrichment, and weighted gene co-expression network analysis were integrated to resolve phenotype-associated transcriptional modules, and hierarchical regulatory layers underlying albinism. Results: CRISPR/Cas9-mediated disruption of pds in potato-generated stable albino phenotypes and revealed extensive transcriptomic reprogramming that was not limited to pigment loss. Albino tissues exhibited more than 9700 differentially expressed genes relative to both wild-type and non-albino edited tissues, whereas non-albino edits showed substantially fewer changes. Functional enrichment demonstrated pervasive suppression of photosynthesis and carbon metabolism alongside activation of secondary metabolism, stress responses, hormone signaling, and cell wall remodeling. WGCNA and cross-validation resolved these changes into distinct, phenotype-associated regulatory layers: MEorangered4 captured coordinated repression of starch and sucrose metabolism (r = −0.998), MEdarkgreen marked albino-linked activation of secondary metabolism and barrier biogenesis (r = 0.855; overlap with Albino Core set, OR = 23.65), while MEblack and MEgrey60 reflected downregulation of stress signaling, proteostasis, and hormone-integrative control and were enriched in transgenic–background-associated gene sets. Conclusions: pds knockout in potato is accompanied by broad transcriptomic changes beyond pigment biosynthesis, suggesting that albinism involves coordinated regulatory and metabolic adjustment under plastid dysfunction rather than pigment loss alone. These results refine the use of pds as a visual editing marker and provide a framework for linking localized genome edits to coordinated network-level transcriptional responses in plants.