In silico design of a SHERLOCK-based point-of-care diagnostic for HIV-1 drug resistance
Creators: name: Ahmed, Armaan
name: De Souza, Diehl
name: Link, Robert
name: Nonnemacher, Michael
name: Wigdahl, Brian
name: Dampier, Will
Publisher: name: Zenodo
Year: 2021
Subjects: HIV-1
Drug resistance
SHERLOCK
CRISPR
LAMP
Point-of-care
Low resource

description: Recent developments in antiretroviral therapy (ART) have reduced human immunodeficiency virus type 1 (HIV-1) infection from a potent killer to a chronic illness. However, major HIV-1 drug resistance mutations (DRMs) hamper ART’s efficacy. Low-resource regions cannot screen for DRMs due to poor infrastructure and high testing costs. To overcome this obstacle, we propose using Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) to act as a rapid and inexpensive HIV-1 DRM diagnostic assay. Here, we present an <em>in silico</em> proof-of-concept using HIV-1 <em>protease</em>. SHERLOCK employs Loop-mediated isothermal AMPlification (LAMP) and a Cas12b-guide RNA (gRNA) complex to isothermally amplify <em>protease</em> sequences and detect any amplified DRM targets, respectively. Different LAMP primers and gRNAs were packaged together to account for HIV-1 genetic variation. DRMs and sequences were collected from the 2019 IAS-USA Drug Resistance Mutations List and Stanford HIV Sequence Database, respectively. LAMP primers were scored by their target diversity and thermodynamic characteristics to select sensitive primer sets. Sequences not targeted by these sets were re-inputted into the pipeline. The best sets from three iterations were combined into a sensitive (70.5%) package. Next, DRM-specific gRNAs were ranked by F<sub>3</sub>-score and the top 128 gRNAs with ≥95% specificity for each DRM were considered for packaging. gRNAs were incrementally added to DRM-specific packages until the following ranked gRNA would only provide an additional sensitivity of &lt;1%. All packages were specific (92.2%±4.3%) and a majority (21/24) were highly sensitive (≥90%). DRM-specific packages were then combined into highly sensitive (95.2%±2.1%) drug-specific meta-packages. Future directions entail improving LAMP primer and gRNA robustness and validating package performances <em>in vitro</em>.
description: This work is supported by NIMH R01 MH110360 (Contact PI, BW), NIMH P30 MH092177 (CNAC/CTRC, Drexel Component PI, BW), NIMH T32 MH079785 (Drexel Component PI, BW)
description: {"references": ["UNAIDS. UNAIDS data 2020. 1\u2013432 https://www.unaids.org/en/resources/documents/2020/unaids-data (2020).", "WHO. Update of recommendations on first- and second-line antiretroviral regimens. 1\u201315 https://www.who.int/hiv/pub/arv/arv-update-2019-policy (2019).", "Wensing, A. M. et al. 2019 update of the drug resistance mutations in HIV-1. Topics in Antiviral Medicine 27, 111\u2013121 (2019).", "WHO. Global action plan on HIV drug resistance 2017\u20132021. (2017).", "Clutter, D. S., Jordan, M. R., Bertagnolio, S. &amp; Shafer, R. W. HIV-1 drug resistance and resistance testing. Infection, Genetics and Evolution 46, 292\u2013307 (2016).", "Hamers, R. L., Sigaloff, K. C. E., Kityo, C., Mugyenyi, P. &amp; de Wit, T. F. R. Emerging HIV-1 drug resistance after roll-out of antiretroviral therapy in sub-Saharan Africa. Curr. Opin. HIV AIDS 8, (2013).", "Joung, J. et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv 2020.05.04.20091231 (2020) doi:10.1101/2020.05.04.20091231.", "Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O. &amp; Zhang, F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat. Protoc. 14, 2986\u20133012 (2019).", "Teng, F. et al. CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity. Genome Biol. 20, 132 (2019).", "Li, L. et al. HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation. ACS Synth. Biol. 8, 2228\u20132237 (2019).", "Curtis, K. A., Rudolph, D. L. &amp; Owen, S. M. Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP). J. Virol. Methods 151, 264\u2013270 (2008).", "Ocwieja, K. E. et al. A Reverse Transcription Loop-Mediated Isothermal Amplification Assay Optimized to Detect Multiple HIV Subtypes. PLOS ONE 10, e0117852 (2015)."]}