Gene editing of human CCR5 by an enhanced CRISPR/SaCas9 nickase system
Date
2017
Authors
Hanson, Britt
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Abstract
The human immunodeficiency virus (HIV) infects 37 million people and causes acquired immune deficiency syndrome (AIDS) that claims the lives of millions annually. Combination antiretroviral therapy has significantly reduced morbidity and mortality rates but latent viral reservoirs established during acute infection preclude drug- and immune-mediated clearance, necessitating lifelong treatment. HIV type 1 (HIV-1) entry is mediated by the interaction with human cluster of differentiation 4 and a co-receptor, mainly C-C chemokine receptor 5 (CCR5) or alternatively C-X-C chemokine receptor 4. A naturally occurring 32 base pair deletion in the CCR5 gene (CCR5-Δ32) renders the receptor non-functional and sequesters native CCR5 in intracellular compartments. CCR5-Δ32 homozygotes have no known immune abnormalities and are resistant to R5-tropic (CCR5-utilising) HIV-1 infection while heterozygotes display reduced infectivity and prolonged progression to AIDS. CCR5 knock out has thus emerged as a promising model for HIV-1 functional cure development. Highly specific targeted genome engineering has provided the means to selectively and permanently manipulate the genetic code that governs all cellular processes. CRISPR/Cas9 is a novel versatile and powerful gene editing tool that employs a short programmable single guide RNA (sgRNA) for delivery of the Cas9 endonuclease to the desired DNA target site, inducing formation of a double stranded break and insertion, deletion and substitution mutations via the error-prone non-homologous end joining DNA repair pathway. The Staphylococcus aureus derived CRISPR/Cas9 system is AAV vector deliverable and has a high level of on target specificity. This system, as well as nickase and „enhanced specificity‟ variants, were employed for disruption of the human CCR5 gene in cell culture. The cleavage activity of ten sgRNAs with the nuclease and five sgRNA pairs with the nickase were assessed for cleavage activity using the T7EI, TIDE and ddPCR™ assays. Deep sequencing was carried out to characterise nuclease and nickase-mediated indels and assess off target activity of two sgRNAs at five highly similar genomic target sites each. The SaCas9 cleavage efficiency varied across sgRNA target sites and was reduced by the nickase and „enhanced specificity‟ modifications, suggesting a trade-off between on target specificity and cleavage efficiency. Finally the effect of CCR5 target site indels on mRNA levels and R5-tropic HIV-1 pseudovirus infectivity was assessed by qRT-PCR and a TZM-bl luciferase assay respectively to determine the functional effect of gene editing. The reason for the observed reduction in mRNA levels was inconclusive, however, the desired phenotypic effect of preclusion of R5-tropic HIV-1 infection was achieved using the WT SaCas9 nuclease,
nickase and „enhanced specificity‟ nuclease systems. This study contributes towards understanding the functionality of novel highly specific CRISPR/Cas9 variants with the capacity to be delivered as a gene therapy both in vivo and ex vivo using AAV vectors for the functional cure of HIV-1.
Description
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine by dissertation
Johannesburg, November 2017.