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. 2019 Jun;2(3):165-171.
doi: 10.1089/crispr.2019.0011.

Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13

Omar O Abudayyeh  1   2   3   4   5 , Jonathan S Gootenberg  1   2   3   4   6 , Max J Kellner  1 , Feng Zhang  1   2   3   4
Affiliations

Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13

Omar O Abudayyeh et al. CRISPR J. 2019 Jun.

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.

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Conflict of interest statement

J.S.G., O.O.A, M.J.K., and F.Z. are co-inventors on patent applications filed by the Broad Institute relating to work in this study. J.S.G., O.O.A., and F.Z. are co-founders of Sherlock Biosciences. F.Z. is a co-founder and advisor of Beam Therapeutics, Editas Medicine, Pairwise Plants, and Arbor Biotechnologies. O.O.A. and J.S.G. are advisors for Beam Therapeutics. J.S.G. is a campus advisor of Benchling, Inc.

Figures

<b>FIG. 1.</b>
FIG. 1.
Rapid soybean gene detection with SHERLOCK. (A) A schematic of SHERLOCK in combination with a rapid genomic DNA extraction method allowing for detection of soybean genes in a quantitative, multiplexed, and portable manner via lateral flow strips. (B) Detection of the CP4 EPSPS gene using SHERLOCK and LwaCas13a in glyphosate-resistant (GR) soybeans and wild type (WT) soybeans over time. (C) SHERLOCK detection of the CP4 EPSPS gene and a positive control gene LE1 using LwaCas13a and a fluorescent reporter.
<b>FIG. 2.</b>
FIG. 2.
Quantitative nucleic acid detection from soybean extract with SHERLOCK. (A) SHERLOCK signal detection of the CP4 EPSPS gene from soybean mixtures containing varying amounts of GR soybeans at the 30 min time point. (B) Quantitative SHERLOCK detection of the percent of the CP4 EPSPS gene in a complex mixture of soybeans (R = 0.98).
<b>FIG. 3.</b>
FIG. 3.
Multiplexed detection of two genes from soybean extracts with SHERLOCK. (A) Schematic of in-sample multiplexed detection of CP4 EPSPS and LE1 genes using two-color SHERLOCK with LwaCas13a and PsmCas13b. (B) In-sample multiplexed detection of the CP4 EPSPS and LE1 genes using two-color SHERLOCK with LwaCas13a and PsmCas13b. LE1 detection of soybeans is compared to a no input water control sample.
<b>FIG. 4.</b>
FIG. 4.
Portable gene detection from soybean extracts with SHERLOCK. (A) Schematic of rapid soybean nucleic acid detection using SHERLOCK lateral flow strips. (B) Rapid detection of the CP4 EPSPS gene within 30 min on lateral flow strips using SHERLOCK and LwaCas13a. (C) Quantitation of the sample band intensities from the lateral flow strips in g.
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