CRISPR-Cas12a fluorescence assays identify weedy Amaranthus species.

Tall Waterhemp (Amaranthus tuberculatus) and Palmer Amaranth (Amaranthus palmeri) populations have developed multiple-herbicide resistance and impact the production of important crops, including corn, soybean and cotton. Morphological plasticity of these species, their persistence in the soil seed bank and their presence as contaminants during trade require efficient and sensitive methodologies to support their identification. Cas12 enzymes can be directed to a specific genomic region by a crRNA, cutting the DNA double strand and exhibiting collateral enzymatic activity on free-floating single-stranded DNA. This characteristic can be used to generate single-stranded oligonucleotide reporters bearing a fluorophore and a quencher, which will produce fluorescence when cut by Cas12. We designed CRISPR-Cas12 fluorescent assays to differentiate A. tuberculatus, A. palmeri and A. palmeri`s sister species (A. watsonii) from other Amaranthus species. Our assays for identifying the A. palmeri + A. watsonii clade and A. tuberculatus were 100% accurate when presented with blind samples of 14 Amaranthus species. Fluorescence could be detected using a blue light filter on a transilluminator within minutes. A preliminary recombinase amplification step to increase the limit of detection, and the Cas12 reaction, can be performed at room temperature and within an hour once DNA has been isolated.