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

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.

Fig. 1

Overview of CRISPR-Cas 12 based assay approaches. Firstly, a crRNA is complexed with a Cas12 enzyme in reaction buffer. Secondly, reporter oligos and sample RT-RPA or RT-PCR product are introduced to the reaction buffer. Thirdly, target sequence recognition causes enzyme activation and collateral cleavage of reporter ssDNA. Lastly, reporter molecules either FAM-Biotin or FAM-Quencher are respectively reported on via lateral flow or Fluorescence Assay

Fig. 2

Benchmarking of varied hardware setups, configuration indicated on the left. Thread configuration indicated in bar as (thread count x). All tested configurations and the average runtime of the tool in hours dependent on core count with thread count indicated in white

Fig. 3

Generated primer pairs from PrimedSherlock alongside background genomic sequences and target sequences were utilized as input for PrimedSherlock. Consensus amplicons were generated to determine valid crRNA targets specific to each virus. Discovered crRNA targets were analyzed for strain coverage and potential off target recognition. Scoring was performed to determine the best two primer & crRNA pairs

Fig. 4

Genomic DNA from Aedes aegypti was spiked with virus genomic RNA. Samples were processed to inactivate and extract viral RNA. RT-PCR as well as RT-RPA was performed. Amplified cDNAs were utilized as templates for Cas12 fluorescence detection assays

Fig. 5

A.s CRISPR-Cas12a was used to validate PrimedSherlock crRNA targets. Fluorescence detection assays for top-ranked primer and crRNA combinations. Fluorescence values represent the average of two technical replicates measured every minute for two hours. Control assays were conducted without virus gRNA template (No Template) and for each strain without Reverse Transcriptase (-RT)

Fig. 6

CRISPR-Cas12a Ultra was used to further validate PrimedSherlock crRNAs. Results of fluorescence detection assays for top-ranked primer and crRNA combinations. Fluorescence values represent the average of two biological replicates measured every minute for two hours. Controls assays were conducted without virus gRNA template (No Template), with two non-target strains per sample (Off-Target Flaviviruses), and for target samples without Reverse Transcriptase (-RT)