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.

Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a

“…Additionally, the developed Cas12a systems are almost unable to achieve single nucleotide specificity for ssDNA targets . There is great interest in structural modification of easily synthesized and programmable crRNA to improve the specificity and efficacy of Cas12a systems. − The crRNA structures can impact not only binding affinity of Cas12a nuclease to target DNA but also the processing performance of Cas12a systems. For example, adding an extended 7 nt DNA sequence onto the crRNA can exponentially elevate the sensitivity and specificity of Cas12a systems for nucleic acid assay .…”

Latest Advances in Arbovirus Diagnostics

“…RPA has been coupled with the CRISPR-Cas12 system and a lateral flow or fluorescent read-out for the diagnosis of West Nile virus, Zika and all four dengue subtypes [ 86 ].…”

“…These assays have been paired with INAAT, such as LAMP, RPA, HDA and SDA, to produce assays that are cheap, deployable in the field and suitable for POC applications. To date, the CRISPR-Cas-12/13 systems have been used for the detection of several important arboviruses, including dengue, West Nile virus and Zika [ 86 ], Japanese encephalitis virus [ 120 ], Crimean-Congo hemorrhagic fever virus [ 121 ] and severe fever with thrombocytopenia symptoms virus [ 122 ].…”

Advancements in Airborne Viral Nucleic Acid Detection with Wearable Devices

“…[2,41,42] The complementary base pairing of nucleic acid sequences through Watson-Crick interactions are conducive to highly specific binding interactions, with probes being able to discriminate between closely related sequences. [41,43,44] However, since target analyte recognition through binding cannot be directly monitored, it needs to be transduced into a user-readable format. This occurs during the final stage of viral RNA or DNA detection, and reporting, with analyte detection being commonly transduced into a user-interpretable colorimetric or fluorescent signal.…”