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Design of a SHERLOCK-based low resource screening assay for HIV-1 drug resistance
Conference poster

Design of a SHERLOCK-based low resource screening assay for HIV-1 drug resistance

Armaan Ahmed, Diehl R. De Souza, Robert W. Link, Michael R. Nonnemacher, Brian Wigdahl and Will Dampier
21 Oct 2021
DOI: https://doi.org/10.5281/zenodo.5719853
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Abstract

HIV-1 CRISPR Low resource SHERLOCK LAMP Point-of-care Drug Resistance
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 are less capable of screening for DRMs due to poor infrastructure and high testing costs. To overcome this, we propose that Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) can be used for rapid and inexpensive HIV-1 DRM diagnosis. SHERLOCK employs Loop-mediated isothermal AMPlification (LAMP) and a gRNA complex to isothermally amplify protease sequences and detect any amplified DRM targets, respectively. To account for HIV-1 genetic diversity, LAMP primers and gRNA packages were generated to ensure robust amplification and cleavage. A LAMP package with relatively high predicted sensitivity (66.9%) was generated by scoring each primer by their target diversity and thermodynamic characteristics and then combining the top-performing primer sets. Next, DRM-specific gRNAs were ranked by F3-score and the top predicted sensitive and specific gRNAs for each DRM were considered for packaging. All gRNA packages demonstrated high predicted specificity (92.2% 4.3%), and 21/24 demonstrated high predicted sensitivity ( 90%). DRM-specific packages were then combined into drug-specific meta-packages with high predicted sensitivity (95.2% 2.1%). Upon in vitro validation, one of our generated LAMP primer sets amplified 13/14 patient-derived HIV-1 isolates and outperformed a published protease-specific LAMP set, and our gRNA showed some ability to cleave targets. Future directions entail improving LAMP primer and gRNA robustness and performing further in vitro validation. This work is supported by NIMH R01 MH110360 (Contact Multi-PI, BW), NIMH P30 MH092177 (CNAC/CTRC, Drexel Component PI, BW), NIMH T32 MH079785 (Drexel Component PI, BW) {"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. & 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. & 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. & 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. & 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).", "Armaan Ahmed. (2021). ArmaanAhmed22/TaskMessenger: TaskMessenger v1.0.0 (v1.0.0). Zenodo. https://doi.org/10.5281/zenodo.5684676"]}

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