Viral diversity is an obligate consideration in CRISPR/Cas9 designs for targeting the HIV reservoir

BMC Biology
Pavitra RoychoudhuryKeith R Jerome

Abstract

RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV. We identified a set of gRNAs targeting HIV LTR, gag, and pol using publicly available sequences for these genes and ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally available sequences. We then selected 59 gRNAs from our list of highly conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR...Continue Reading

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Citations

Jul 10, 2020·AIDS Research and Human Retroviruses·Katherine J SessionsAlexandra L Howell
Aug 20, 2020·Nature Communications·Martine AubertKeith R Jerome
Jan 9, 2019·Frontiers in Microbiology·Amanda R PanfilKristine E Yoder
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Dec 23, 2020·Human Gene Therapy·Tatjana I CornuToni Cathomen
Feb 16, 2021·The Journal of Infectious Diseases·Yijia LiJonathan Z Li
Jun 30, 2021·Journal of the Royal Society, Interface·Daniel B ReevesBryan T Mayer

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Methods Mentioned

BETA
transfection
flow cytometry

Software Mentioned

Geneious
lmerTest
LANL
OpenCyto
Bioconductor
Galaxy
R
Trimmomatic
Bowtie2

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