HIV-1 promoter is gradually silenced when integrated intoBACH2in Jurkat T-cells

Abstract

Background The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). Methods We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean–/mCherry+) HIV-1 promoters were characterised. Results Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Conclusion Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.

Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation

“…We generated a barcoded version of our previously described HIV-1-based dual-fluorophore vector LTatCL[M] 25 , 26 to transfect Jurkat T cells and integrate the vector in the GSH AAVS1 site in the human genome via CRISPR-Cas9. Targeted insertion of our HIV-1-based dual-fluorophore vector LTatCL[M] into the GSH AAVS1 was extensively studied in our previous study, showing continuous transgene expression for up to 160 days in cell populations.…”

“…The vector is flanked by AAVS1 homologous arms in two ways to integrate the vector in the same (s) and convergent (c) transcriptional orientation, respectively, relative to the host gene PPP1R12C ( Figure 1 A). The original vector LTatCL[M] 25 was developed primarily to study HIV-1 latency. The new vector LTatC10NL[M], therefore, contains a second fluorophore (mCherry) driven independently of the HIV-1 promoter by the constitutive heIF4A1 promoter and flanked by two insulators ( Figure 1 A).…”

“…They consist of two reporter genes, Cerulean under the control of the HIV-1 promoter and mCherry, which is driven by the constitutive human elongation initiation factor 4A1 (heIF4A1) promoter and flanked by two insulators. 25 , 26 Targeted insertion of our HIV-1-based dual-fluorophore vector LTatCL[M] via CRISPR-Cas9 into the GSH AAVS1 in the same and convergent orientation showed continuous expression of both reporter genes for up to 160 days. 25 …”

“… 25 , 26 Targeted insertion of our HIV-1-based dual-fluorophore vector LTatCL[M] via CRISPR-Cas9 into the GSH AAVS1 in the same and convergent orientation showed continuous expression of both reporter genes for up to 160 days. 25 …”

“…To further characterize the GSH AAVS1, we examined the stochastic fluctuations upon insertion of a barcoded transgene into the GSH AAVS1. We first modified our HIV-1-based dual-fluorophore vector LTatCL[M] 25 by inserting a barcode consisting of 10 random nucleotides. We then inserted the vector LTatC10NL[M] into the GSH AAVS1 in Jurkat T cells via CRISPR-Cas9 in the same and convergent orientation relative to the host gene and studied the barcode expression.…”