Design of a SHERLOCK-based low resource screening assay for HIV-1 drug resistance
Creators: name: Ahmed, Armaan
name: De Souza, Diehl R.
name: Link, Robert W.
name: Nonnemacher, Michael R.
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 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 <em>protease </em>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 <em>in vitro </em>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 <em>in vitro </em>validation.
description: 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)
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