An RT-RPA–CRISPR/Cas12a-based assay for robust sexual Identification of dioecious plants

Abstract

Dioecious plants, like Date palm (Phoenix dactylifera) and Jojoba (Simmondsia chinensis), are a type of plant that
do not have sexual identification in the same way that animals or humans do. Sexual identification in plants
typically involves determining the sex of individual plants within a dioecious species. Date palms and jojoba are
dioecious, with male and female trees, and their sexual identification is important in agriculture for proper
pollination, but until now it doesn't involve methods like RPA (Recombinase Polymerase Amplification) or
CRISPR. We successfully developed a CRISPR/Cas12a-based fluorescence test for the sexual identification of
date palm and jojoba as models for dioecious, with RPA and CRISPR-Cas12a, which could be used as a simple,
accurate, and fast detection method in agricultural fields due to its advantages of high-sensitivity. This
investigation uses our proprietary technology to detect sub-kilobase non-repetitive genomic loci, CRISPR-FISH.
In this study, we designed 16 different sgRNAs using full-length Human SRY as the substitute target in date palm
and jojoba, given the extremely high homology between Human SRY and Date Palm SRY gene. These sgRNAs
were labeled them 3 Atto565 dyes per sgRNA to visualize them at a single-cell level. Fluorescence in situ
hybridization (FISH) has been employed as a potent and practical method for directly identifying certain DNA
segments inside the genome. In this method, plant chromosomes are physically mapped using ribosomal DNA
genes (45S and 5S rDNA) as markers to examine genomic organization. The initial result for SRY staining in a
substitute cell line, the Human A549 cell, a male cell line containing a single copy of SRY was promising albeit
with high background staining. Prominent dots (red) in the nuclei region were observed, but with high background
staining in both nuclei and cytosol. This refined technique is perfect for quickly localizing tiny DNA segments
and single-copy genes. Additionally, PRINS is a quicker and less expensive option than FISH. Furthermore, the
successful application of the assay for Sexual identification of some Dioecious plant samples highlighted its
potential for rapid and accurate sex detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a
diagnostic method offers a potentially valuable technological solution and management for Dioecious plant sexdetermination at very early stage.