Shoppell, E. A. | bioRxiv Science

bioRxiv2025

Glow with the Flow: Reproducible Analysis of Transiently Transformed Protoplasts Using Dual Fluorescent Reporters in R

Transient transformation assays using protoplasts have become a widely employed technique in plant research. Positive fluorescent selection was subsequently developed to assess the effect of transient effector gene expression in only successfully transfected cells using flow cytometry. This process, though effective, often requires considerable manual effort and subjective judgment to quantify reporter gene expression in the intended cell populations. To address this, we introduce a new, open-source workflow based on the R programming language. This method enhances the reproducibility and scalability of such experiments, which enable rapid study of gene regulation and signal transduction in plants. This workflow is available at https://github.com/PlantSynBioLab/positive-fluorescence-selection.

plant biology↗

bioRxiv2024

Optimized CRISPR inhibition and activation opens key avenues for systematic biological exploration in zebrafish.

The application of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. We report proof-of-principle data evaluating transient expression of a codon optimized CRISPRi/a system for zebrafish across established pigmentary and growth phenotypes. A codon-optimized and catalytically inactive cas9 gene (dcas9) was cloned upstream of codon-optimized Kruppel associated box (KRAB) and methyl-CpG binding protein 2 (MeCP2) for CRISPRi, and VP64 for CRISPRa. To validate CRISPRi, we targeted key genes in melanocyte differentiation (sox10 and mitfa); and melanin production (tyrosinase; tyr). Microinjection of CRISPRi mRNA and single guide RNAs (sgRNAs) targeting the tyr promoter or 5-UTR resulted in larvae with hypopigmented epidermal melanocytes. Similarly, CRISPRi targeting of the sox10 or mitfa promoters results in hypopigmentation of epidermal melanocytes consistent with their roles upstream of tyr, and the role of sox10 in activation of mitfa. Finally, we tested both CRISPRi/a to modulate a single gene to yield hypomorphic and hypermorphic effects, selecting mrap2a as our target. This gene regulates energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene (mc4r). We show that inactivating or activating mrap2a with CRISPRi/a significantly decreases or increases larval body length, respectively. We demonstrate that CRISPRi/a can modulate control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.

genetics↗