Abstract
Background: CRISPR-Cas based diagnostic assays provide a portable solution which bridges the benefits of qRT-PCR and serological assays in terms of portability, specificity and ease of use. CRISPR-Cas assays are rapidly fieldable, specific and have been rigorously validated against a number of targets, including HIV and vector-borne pathogens. Recently, CRISPR-Cas12 and CRISPR-Cas13 diagnostic assays have been granted FDA approval for the detection of SARS-CoV-2. A critical step in utilizing this technology requires the design of highly-specific and efficient CRISPR RNAs (crRNAs) and isothermal primers. This process involves intensive manual curation and stringent parameters for design in order to minimize off-target detection while also preserving detection across divergent strains. As such, a single, streamlined bioinformatics platform for rapidly designing crRNAs for use with the CRISPR-Cas12 platform is needed. Here we offer PrimedSherlock, an automated, computer guided process for selecting highly-specific crRNAs and primers for targets of interest.
Results: Utilizing PrimedSherlock and publicly available databases, crRNAs were designed against a selection of Flavivirus genomes, including West Nile, Zika and all four serotypes of Dengue. Using outputs from PrimedSherlock in concert with both wildtype A.s Cas12a and Alt-R Cas12a Ultra nucleases, we demonstrated sensitive detection of nucleic acids of each respective arbovirus in in-vitro fluorescence assays. Moreover, primer and crRNA combinations facilitated the detection of their intended targets with minimal off-target background noise.
Conclusions: PrimedSherlock is a novel crRNA design tool, specific for CRISPR-Cas12 diagnostic platforms. It allows for the rapid identification of highly conserved crRNA targets from user-provided primer pairs or PrimedRPA output files. Initial testing of crRNAs against arboviruses of medical importance demonstrated a robust ability to distinguish multiple strains by exploiting polymorphisms within otherwise highly conserved genomic regions. As a freely-accessible software package, PrimedSherlock could significantly increase the efficiency of CRISPR-Cas12 diagnostics. Conceptually, the portability of detection kits could also be enhanced when coupled with isothermal amplification technologies.
Keywords: CRISPR-Cas12; Cas12 nuclease assay; Cas12a; Dengue; Field forward diagnostics; PrimedRPA; PrimedSherlock; Sherlock; Vector-borne disease; West Nile; Zika.
【저자키워드】 SHERLOCK, Dengue, West Nile, Cas12a, CRISPR-Cas12, Zika., Vector-borne disease, PrimedSherlock, PrimedRPA, Field forward diagnostics, Cas12 nuclease assay, 【초록키워드】 SARS-CoV-2, HIV, qRT-PCR, bioinformatics, polymorphism, diagnostic, diagnostics, RNA, CRISPR, nucleic acid, specificity, Serological assay, target, Pathogens, automated, Zika, isothermal amplification, targets, genomes, flavivirus, genomic, Strains, Intensive, Critical, platform, Combination, strain, isothermal, Efficiency, In-vitro, serotype, FDA approval, wildtype, primer, West, Primers, manual curation, nucleases, offer, parameter, regions, field, benefit, robust, conserved, significantly, assays, facilitated, demonstrated, primer pair, CRISPR-Ca, Nile,