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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.

SUBMITTER: Li L

PROVIDER: S-EPMC8713540 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The efficiency of the CRISPR-Cas system is highly dependent on well-designed CRISPR RNA (crRNA). To facilitate the use of various types of CRISPR-Cas systems, there is a need for the development of computational tools to design crRNAs which cover different CRISPR-Cas systems with off-target analysis capability. Numerous crRNA design tools have been developed, but nearly all of them are dedicated to design crRNA for genome editing. Hence, we developed a tool matching the needs of both beginners and experts, named CaSilico, which was inspired by the limitations of the current crRNA design tools for designing crRNAs for Cas12, Cas13, and Cas14 CRISPR-Cas systems. This tool considers a comprehensive list of the principal rules that are not yet well described to design crRNA for these types. Using a list of important features such as mismatch tolerance rules, self-complementarity, GC content, frequency of cleaving base around the target site, target accessibility, and PFS (protospacer flanking site) or PAM (protospacer adjacent motif) requirement, CaSilico searches all potential crRNAs in a user-input sequence. Considering these features help users to rank all crRNAs for a sequence and make an informed decision about whether a crRNA is suited for an experiment or not. Our tool is sufficiently flexible to tune some key parameters governing the design of crRNA and identification of off-targets, which can lead to an increase in the chances of successful CRISPR-Cas experiments. CaSilico outperforms previous crRNA design tools in the following aspects: 1) supporting any reference genome/gene/transcriptome for which an FASTA file is available; 2) designing crRNAs that simultaneously target multiple sequences through conserved region detection among a set of sequences; 3) considering new CRISPR-Cas subtypes; and 4) reporting a list of different features for each candidate crRNA, which can help the user to select the best one. Given these capabilities, CaSilico addresses end-user concerns arising from the use of sophisticated bioinformatics algorithms and has a wide range of potential research applications in different areas, especially in the design of crRNA for pathogen diagnosis. CaSilico was successfully applied to design crRNAs for different genes in the SARS-CoV-2 genome, as some of the crRNAs have been experimentally tested in the previous studies.

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

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