Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

Frontiers in Cellular and Infection Microbiology

Will Dampier¹  Alexander G. Allen¹  Andrew Atkins¹  Robert W. Link¹  Cheng-Han Chung¹  Brian Wigdahl²  Michael R. Nonnemacher² 

[1] Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States;Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States;Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States;Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States;Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, United States;

关键词: human immunodeficiency virus type 1 (HIV-1);    CRISPR/Cas9;    gene therapy;    genetic variation;    HIV-1 subtypes;    bioinformatics;    gRNA design;   

DOI  :  10.3389/fcimb.2021.593077

来源: Frontiers

【 摘 要 】

Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites.

【 授权许可】

CC BY