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

• Published in: BMC bioinformatics Vol. 23; no. 1; p. 428
• Main Authors: Mann, James G, Pitts, R Jason
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 14-10-2022; BioMed Central; BMC
• Subjects: Assaying; Automation; Background noise; Bioinformatics; Cas12a; Collateral; Computational biology; Computer-aided medical diagnosis; COVID-19; CRISPR; CRISPR-Cas Systems - genetics; CRISPR-Cas12; Dengue fever; Design; Design parameters; Divergence; Enzymes; FDA approval; Field forward diagnostics; Fluorescence; Genomes; HIV; Human immunodeficiency virus; Humans; Identification and classification; Medical importance; Methods; Molecular diagnostic techniques; Mutation; Nuclease; Nucleic Acids; Portability; PrimedRPA; PrimedSherlock; Primers (Molecular genetics); RNA; SARS-CoV-2 - genetics; Serotypes; Severe acute respiratory syndrome coronavirus 2; Sherlock; Target detection; Vector-borne diseases; West Nile virus; Zika virus; Zika Virus - genetics; Zika Virus Infection; United States; CRISPR-Cas12; Sherlock; Field forward diagnostics; PrimedSherlock; Dengue; West Nile; Cas12 nuclease assay; Cas12a; PrimedRPA; Vector-borne disease; Zika
• ISSN: 1471-2105; 1471-2105
• DOI: 10.1186/s12859-022-04968-5

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.
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.
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.