Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13

Abstract

Nucleic acid detection is vital for agricultural applications including trait detection during breeding, pest surveillance, and pathogen identification. Here, we use a modified version of the CRISPR-based nucleic acid detection platform SHERLOCK to quantify levels of a glyphosate resistance gene in a mixture of soybeans and to detect multiple plant genes in a single reaction. SHERLOCK is rapid (*15 min), quantitative, and portable, and can process crude soybean extracts as input material for minimal nucleic acid sample preparation. This field-ready SHERLOCK platform with color-based lateral flow readout can be applied for detection and quantitation of genes in a range of agricultural applications.

CRISPR-Cas13d mediates robust RNA virus interference in plants

“…Intriguingly, these systems, once activated through the specific binding of the target RNA, confer collateral "promiscuous" activity and non-specifically degrade the RNA transcripts in the cell [24]. This collateral activity was harnessed to develop highly sensitive pathogen detection methods [28][29][30][31].…”

CRISPR/Cas13a‐Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification‐Free miRNA Diagnostics

“…These two specific characteristics of the Cas enzyme enable a highly selective and sensitive detection. So far, Cas13a was used for the optical detection of virus RNAs or even for the detection of plant genes, whereas no research work exists up to now, combining CRISPR/Cas13a with an electrochemical detection method for miRNA diagnostics . Here, we present the CRISPR technology on a microfluidic electrochemical biosensor for measuring miRNA levels of the potential brain tumor marker miR‐19b in serum samples from patients, suffering from brain cancer.…”

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine

“…The SHERLOCK assay has been successfully used to detect both Zika virus and dengue virus directly from bodily fluids, including urine and respiratory samples, in less than two hours and enabled discrimination among viral serotypes [29,44]. This approach may soon extend to serum samples, and has potential for the rapid diagnosis of human immunodeficiency virus (HIV) as well as hepatitis c virus (HCV) genotype in order to guide the choice of antiviral therapy, which may depend on the presence of single nucleotide polymorphisms [45][46][47][48].…”

“…For now, CRISPR-based clinical research will focus on the identification and alteration of infectious pathogens as well as novel treatment options for a variety of diseases ranging from infections to heart disease and cancer [45,[100][101][102][103].…”