Biology subjects

Cody, W. B.

Publications and source records attributed to Cody, W. B..

3 recordsLinked to original sources

bioRxiv2025

An in planta single-cell screen to accelerate functional genetics

Genetic screens in whole plants are a powerful tool for functional genetics. However, elucidating gene function in highly redundant genetic programs such as signaling pathways remains challenging in both model and non-model plants. Here, we report a single-cell screening platform, PIVOT (Protoplast Isolation after Virus Overexpression in planTa), to accelerate identification and functional characterization of plant genes. We used Nicotiana benthamiana as a heterologous host to test gene libraries arrayed in a single leaf. Two elements of our system made pooled screens possible in planta: (1) we harnessed viral superinfection exclusion to ensure single multiplicity of infection per cell during pooled library delivery, and (2) we engineered a cell surface protein as a phenotypic marker for isolating cells of interest from a heterogeneous population. Using this system, we recovered known and new regulators of cytokinin signaling from an Arabidopsis open reading frame library. We anticipate PIVOT will be broadly applicable for high-throughput, single-cell functional genetic screening across the plant kingdom.

plant biology↗

bioRxiv2022

Modular and programmable RNA sensing using ADAR editing in living cells

With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/stats in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular and programmable design for live RNA sensing using adenosine deaminases acting on RNA (RADAR). We validated and then expanded our basic design, characterized its performance, and thoroughly analyzed its compatibility with the human/mouse transcriptomes. We also identified strategies to further boost output levels and improve the dynamic range. We show that RADAR is programmable and modular, and uniquely enables compact AND logic. In addition to being quantitative, RADAR can distinguish disease-relevant point mutations. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.

bioengineering↗

bioRxiv2020

Native ribonucleases process sgRNA transcripts to create catalytic Cas9/sgRNA complexes in planta

The current CRISPR/Cas9 gene editing dogma for single guide RNAs (sgRNA) delivery is based on the premise that 5' and 3' nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo. This has led to engineering strategies designed to either avoid or remove extraneous nucleotides on the 5' and 3' termini. Previously, we used a Tobacco mosaic virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana benthamiana. This vector yielded high levels of GFP and catalytically active sgRNAs. Here, in an effort to understand the biochemical interactions of this result, we used in vitro assays to demonstrate that nucleotide overhangs 5', but not 3', proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at the specified target site. Next we showed that in planta sgRNAs bound to Cas9 are devoid of the expected 5' overhangs transcribed by the virus. Furthermore, when a plant nuclear promoter was used for expression of the GFP-sgRNA fusion transcript it also produced indels when delivered with Cas9. These results reveal that 5' "auto-processing" of progenitor sgRNAs occurs natively in plants. Towards a possible mechanism for the perceived "auto-processing", we found, using in vitro generated RNAs and those isolated from plants, that the 5' to 3' exoribonuclease XRN1 can degrade elongated progenitor sgRNAs whereas the mature sgRNA end-products are resistant. Comparisons with other studies suggest that sgRNA "auto-processing" may be a phenomenon not unique to plants, but other eukaryotes as well.

plant biology↗