A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

Abstract

For some Cas nucleases, trans -cleavage activity triggered by CRISPR/Cas-mediated cis -cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field. Supporting Information The supporting information is available online at 10.1007/s11427-021-2028-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

CRISPR/Cas-Based Diagnostics in Agricultural Applications

“…For multiplex reaction, FAM labeled ssDNA and HEX labeled ssRNA reporters were used with LbCas12a and LwCas13a in the same reaction to detect FV and FG together. In the presence of target, LbCas12a preferentially cleaves ssDNA reporters and LwCas13a cleaves ssRNA reporters, which generates two types of fluorescence signal with different wavelengths when excited with a portable fluorescence flashlight (i.e., LUYOR-3415RG) . This developed technology is similar to the improved SHERLOCK platform reported by Gootenberg et al (Figure B).…”

“…For visual observation of FAM, a 488 nm excitation filter was utilized, while a 520 nm filter was used for HEX, with yellow and red glasses, respectively. Furthermore, to make the system field-applicable, a portable solar generator and dry bath incubator were employed for DNA crude extract preparation and RPA amplification . With this method a single target was detected in 30 min, while a multiplex sample required 60 min.…”

PrimedSherlock: A tool for rapid design of highly specific CRISPR-Cas12 crRNAs

“…In terms of crRNA specificity for target sequences, as few as two mismatches can cause a significant reduction in detection efficiency [24, 25, 26]. Furthermore, mismatches in the seed region, or bases 1-6 proximal to the PAM site, can negatively impact on-target recognition [3, 26, 27, 28].…”

CRISPR-Cas13 in malaria parasite: Diagnosis and prospective gene function identification

“…It is however unknown if this limitation also exists during RNA editing with Cas9. Another peculiar RNA editing advantage of Cas13 is its auto-catalytic ability of Cas13 to process its pre-crRNA, which allows for streamlined delivery of targeting crRNAs for large-scale mRNA transcript editing without loss of specificity and efficiency ( Gootenberg et al, 2018 ; Ackerman et al, 2020 ; Li et al, 2022 ; Thakku et al, 2022 ). This property has already been applied to manipulate a host of genes in live cells ( East-Seletsky et al, 2016 ).…”