Biology subjects

Groover, E. D.

Publications and source records attributed to Groover, E. D..

3 recordsLinked to original sources

bioRxiv2026

Targeted knockout of CYP79A1 reduces cyanogenic potential in grain sorghum

Sorghum bicolor is a climate-resilient C4 crop used for food, forage, and bioenergy, but broader adoption is constrained by accumulation of the cyanogenic glucoside dhurrin, which can release toxic hydrogen cyanide (HCN) upon tissue damage. Dhurrin levels are especially high in juvenile tissues, creating risk for grazing animals and limiting use in mixed crop-livestock systems. Here, we establish a CRISPR-Cas9 genome-editing strategy targeting CYP79A1, which catalyzes the first committed step in dhurrin biosynthesis, in the elite grain sorghum inbred RTx430, yielding transgene-free lines with stable, heritable reduction in cyanogenic potential across early vegetative development. Homozygous cyp79a1 knockouts were negligibly cyanogenic, whereas heterozygous plants exhibited approximately half the cyanogenic potential of unedited nulls. Consistent with established livestock grazing guidelines, only homozygous knockouts fell below thresholds considered hazardous for incidental grazing. This work establishes CYP79A1 as a practical and heritable genome-editing target for reducing sorghum cyanogenesis and provides a clear path for deployment of low-cyanogenic alleles in elite breeding backgrounds.

plant biology↗

bioRxiv2026

Architecting cis-regulation to quantitatively tune gene expression in cereals

Precise modulation of gene expression via cis-regulatory editing holds promise for non-transgenic crop improvement, but the sequence-to-function relationships that govern plant promoter activity remain poorly understood. Here, we develop a massively parallel reporter assay (MPRA) in Sorghum bicolor to systematically measure the effects of >30,000 CRISPR-like mutations-deletions, substitutions, and motif insertions-across entire native promoters and 5' untranslated regions (UTRs) of three photosynthesis genes: PsbS, Raf1, and SBPase. We find that gene expression is most tunable within a [~]500 base pair core promoter region, where mutational effects are reproducible across biological replicates and predictive of protein output. Within these regions, we identify compact deletions and motif insertions that strongly increase protein production (>30-fold relative to wild type), exceeding the performance of transgenic enhancer elements. Mutation-effect relationships are gene-specific, highlighting the need for tailored regulatory maps. Our results establish a high-throughput strategy for cis-regulatory fine-mapping that enables crop improvements via minimal, precise, and non-transgenic gene edits.

synthetic biology↗

bioRxiv2025

Discovery of widespread activating mutations in a compact RNA-guided endonuclease

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the proteins mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

molecular biology↗