HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells

Generation of LTatCL[M] with target locus homologous arms and Cas9/guide RNA-encoding plasmids

In the HIV-1 based, dual-fluorophore vector LTatC[M] the 3′LTR is located downstream of the second fluorophore mCherry to enable retrovirus production and subsequent infection of target cells (Kok et al., 2018). LTatC[M] was modified to LTatCL[M], that is, the 3′LTR (L) was inserted between Cerulean (C) and the insulator cHS4 (Fig. 1A) to further enhance the transcriptional independence of the HIV-1 promoter controlled Cerulean. For targeted integration of this HIV-1 based, dual-fluorophore vector, retrovirus production is not required. Thus, the HIV-1 3′LTR was relocated immediately downstream of Cerulean. Additionally, a polyA signal was inserted between mCherry ([M]) and the second insulator sMAR8 (Fig. 1A). The homologous regions on both sides of the targeted HIV-1 integration site in the human genome were obtained from NCBI GenBank: BACH2 intron 5 (Accession No: NT_007299.13; 5′ arm nucleotides 93′502–94′355, 3′ arm nucleotides 94′356–95′206), BACH2 intron 2 (5′ arm nucleotides 339′363–340′186, 3′ arm nucleotides 340′187–341′034) and AAVS1 (Accession No: NC_000019.10; 5′ arm nucleotides 1′399–2′218, 3′ arm nucleotides 2′219–3′051). Targeted integration sites are depicted in Fig. 1B.

Primers to amplify the respective target locus homologous arms are listed in Table S1. Each PCR reaction contained 100 ng of human genomic DNA (Sup-T1 cell line; obtained through the NIH AIDS Reagent Programme, Division of AIDS, NIAID, NIH, from Dr. Dharam Ablashi), 1x Platinum Taq PCR buffer (ThermoFisher, Waltham, MA, USA), 2 mM MgCl₂ (ThermoFisher, Waltham, MA, USA), 0.2 mM dNTP (NEB), 0.4 μM of each forward and reverse primer and 1 U Platinum Taq polymerase (ThermoFisher, Waltham, MA, USA) in a total volume of 50 μL. The PCR cycling conditions were as follows: 94 °C for 2 min; 35 cycles of (94 °C for 30 s, 55 °C for 30 s, 68 °C for 1 min); 68 °C for 5 min; 4 °C hold.

The respective pair of target locus homologous arms were cloned into pGEM-T Easy (Promega). Subsequently, the dual-fluorophore vector LTatCL[M] was cloned into each plasmid in the same or convergent orientation via blunt-end cloning. The 6 plasmids pBACH2_i5-, pBACH2_i2- and pAAVS1-LTatCL[M] were generated, containing the homologous arms of the targeted HIV-1 integration loci in BACH2 intron 5, BACH2 intron 2, and in the genomic safe harbour AAVS1, respectively, containing LTatCL[M] in both transcriptional orientations (s and c) (Fig. 1A).

Plasmids encoding Cas9 and guide RNAs were based on PX458 (Table S1). pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138; http://n2t.net/addgene:48138; RRID:Addgene_48138) (Ran et al., 2013). Annealing of 100 μM 5′ phosphorylated primers for the guide RNAs was performed using the following conditions: 80 °C for 5 min, 65 °C for 7 min, 60 °C for 7 min, 55 °C for 7 min, 50 °C for 7 min, 45 °C for 7 min, 40 °C for 7 min, 35 °C for 7 min, 30 °C for 7 min, 25 °C for 7 min and 4 °C hold. The annealed guide RNA primers were separately cloned into PX458.

All plasmid sequences were confirmed by next-generation sequencing. Briefly, plasmids were gel purified, diluted to 0.2 ng/μl, and 1 ng of the plasmids were processed using the Illumina Nextera XT DNA library Prep kit. The DNA libraries were subsequently paired-end or single-end sequenced with an Illumina MiSeq using the MiSeq Reagent Kit v3 (150-cycle). Constructs are shown in Fig. 1A.

Transfection of Jurkat T-cells and single cell sorting

Prior to transfection, the six LTatCL[M] vectors (pBACH2_i5-, pBACH2_i5c-, pBACH2_i2s-, pBACH2_i2c-, pAAVS1_s-, and pAAVS1_c- LTatCL[M]) were linearised using 100 units of NsiI (NEB)/20 µg DNA. For transfection, 1 million Jurkat T-cells were resuspended in 100 μL Nucleofector solution (Cell Line Nucleofector^™ Kits, Lonza) and combined with 2 μg of the linearised dual-fluorophore vector and 2 μg of the corresponding gRNA/Cas9 plasmid: PX458_gBACH2^{94,355–94,356} (intron 5), PX458_gBACH2^{340,186–340,187} (intron 2) and PX458_gAAVS1^{2,218–2,219} (AAVS1). Nucleofection was performed using the programme X-001 for Jurkat T-cells (Amaxa^™ Nucleofector^™ II, Lonza). Cells were cultured in RPMI-1640 medium media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (10,000 units/ml penicillin, 10 mg/ml streptomycin).

At day 9 post transfection, cells were analysed using fluorescence-activated cell sorting (FACS) and sorted at 20 cells per well in 96-well plates using a BD FACSAria^™ III (BD Biosciences, Franklin Lakes, NJ, USA). Two cell phenotypes were sorted: 1. Cerulean⁺/mCherry⁺ and 2. Cerulean⁻/mCherry⁺. This was done for each nucleofected cell population: BACH2_i5s, BACH2_i5c, BACH2_i2s, BACH2_i2c, AAVS1_s, and AAVS1_c. After 50 days, expanded cell cultures were analysed by flow cytometry and subsequently single cell sorted. Longitudinal flow cytometric analysis of the cells were done with the LSRFortessa II (BD Biosciences, Franklin Lakes, NJ, USA) and data were analysed using the FlowJo Software v.10.0.8. (FLOWJO, LLC) (Fig. 1C).

Activation of silenced HIV-1 promoter in cells with integrated LTatCL[M] in BACH2

Over time silenced HIV-1 promoter in sorted Cerulean⁺/mCherry⁺ monoclonal cell lines were reactivated using 10 ng/μL tumor necrosis factor alpha (TNF-α) and 4 nM Romidepsin (Ro) (Selleckchem, Houston, TX, USA). After 24 hrs the cells were analysed by flow cytometry.

Amplifying the junctions of LTatCL[M] integration into BACH2 and AAVS1

Genomic DNA was extracted from 5 million transfected Jurkat T-cells using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) and quantified by Quant-iT^™ PicoGreen^™ dsDNA Assay kit (ThermoFisher Scientific, Waltham, MA, USA). To verify the targeted integration of the dual-fluorophore vector LTatCL[M] into BACH2 or AAVS1, the junctions of targeted integration were amplified by (semi-)nested PCR using 300 ng genomic DNA containing 1x PCR buffer (Sigma–Aldrich, St. Louis, MI, USA), 1.5 mM MgCl₂ , 0.2 mM dNTPs, 0.5 μM of each respective forward and reverse primer (Table S1), and 0.5 U JumpStart Taq DNA polymerase (Sigma–Aldrich, St. Louis, MI, USA) in a total volume of 25 μL. The PCR cycling conditions were as follows: 94 °C for 2 min; 35–40 cycles of (94 °C for 30 s, 63 °C for 30 s, 72 °C for 2 min); 72 °C for 5 min; 4 °C hold.

Mono-allelic or bi-allelic integration of LTatCL[M] into BACH2 and AAVS1 was verified by PCR containing 100 ng genomic DNA, 1x PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.5 μM of each respective forward and reverse primers (Table S1), and 0.5 U JumpStart Taq DNA polymerase in a total volume of 25 μL. The PCR cycling conditions were as follows: 94 °C for 2 min; 35 cycles (94 °C for 30 s, 63 °C for 30 s, 72 °C for 2 min); 72 °C for 5 min; 4 °C hold.

Amplicons were sequenced by Sanger sequencing. The sequencing reaction contains 1x Seqmix (Big Dy Termination v1.1, 1x Dilution Buffer (ThermoFisher, Waltham, MA, USA)), and 100 ng DNA in a total volume of 40 μL. 0.2 μM primer was added (Table S1). The PCR cycling condition were as follows: 40 cycles (96 °C for 30 s, 50 °C for 30 s, 60 °C for 4 min); 4 °C hold. Sequencing was performed following the manufacturer’s instruction on an 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Near full-length amplification of integrated vector LTatCL[M]

Genomic DNA was extracted from 3 million transfected cells using the DNeasy Blood and Tissue kit and quantified by Quant-iT^™ PicoGreen^™ dsDNA Assay kit. To investigate the integrity of the integrated vector LTatCL[M], near full-length amplification of the vector was performed, using genomic 300 ng DNA, 1x Long Amp Taq Reaction Buffer (NEB), 0.4 mM dNTPs, 0.5 μM of each forward and reverse primer (Table S1) and 2.5 U Long Amp Taq Polymerase (NEB) in a total volume of 25 μL. The PCR cycling conditions were as follows: 94 °C for 30 min; 30 cycles of (94 °C for 20 s, 58–61 °C for 30 s, 72 °C for 5 min); 72 °C for 10 min; 4 °C hold. A total of 1 ng purified amplicons were processed with the Nextera XT DNA Library Preparation Kit (Illumina) and subsequently sequenced using the MiSeq reagent Kit v2 as described above.

Quantification of BACH2 mRNA expression

RNA was extracted from 3 million transfected cells using the All Prep DNA/RNA kit (Qiagen, Hilden, Germany) and quantified by Nanodrop 1000. A total of 800 ng RNA was reverse transcribed with Prime Script Reverse Transcriptase (Takara, Kyoto, Japan) according to manufacturer’s instructions. To exclude contamination of plasmid and genomic DNA contamination, reactions lacking reverse transcriptase were included for each RNA sample. To quantify the mRNA level of BACH2, qPCR reactions were performed in triplicates for each sample. cDNA was diluted 1:10 mixed with 1x PCR buffer (Sigma–Aldrich, St. Louis, MI, USA), 1.5 mM MgCl₂, 0.2 μM dNTP (NEB), 0.5 μM of each Fw and Rv primers, 1x SYBR^® Green, 50 nM Rox, 0.5 U JumpStart Taq Polymerase (Sigma–Aldrich, St. Louis, MI, USA) in a total volume of 25 μL. For each reaction, primer pair was chosen to amplify a specific region of the BACH2 mRNA, spanning exon 7–8. As a mRNA expression control glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) was amplified with GAPDH-specific primers (Table S1). The qPCR cycling conditions were as follows: 94 °C for 2 min; 40 cycles (94 °C for 30 s, 55 °C for 30 s, 63 °C for 1 min (collect data)); 72 °C for 2 min; 95 °C for 15 s; 4 °C hold. Melt curves were collected to analyse specificity of the amplification. The qPCR was performed in the ABI 7800 real-time PCR thermos-cycler then analysed using the 7500 Software v2.0.4, using the comparative C_t method (Livak & Schmittgen, 2001).

Quantification of BACH2 protein expression

Cells were lysed using assay lysis buffer (50 mM Tris-HCL pH8, 150 mM NaCl, 0.5% Na deoxycholate, 0.5% Triton X-100, 1x Protease inhibitor, ddH₂O) and protein was quantified using the Bicinchoninic acid assay (BCA) (Pierce^™ BCA Protein Assay Kit). A total of 40 μg protein were mixed with loading dye and loaded on a Bolt^™ 4–12% Bis–Tris Plus Gels (ThermoFisher Scientific, Waltham, MA, USA). Subsequently, proteins were transferred onto a nitrocellulose membrane (iBlot^™ 2 Transfer Stacks, ThermoFisher Scientific, Waltham, MA, USA) blocked for 1 h at RT with 5% Top-Block wt/vol in PBS supplemented with Tween-20 (10x PBS pH7.4, 0.1% Tween 20, and ddH₂O). For protein detection, the membrane was incubated with primary antibody (1/400 diluted rabbit anti-BACH2 antibody (PA5-23642; ThermoFisher Scientific, Waltham, MA, USA) and 1/5,000 diluted rabbit-anti-GAPDH antibody (ab9485, Abcam, Cambridge, UK)) overnight at 4 °C. After washing the membrane four times with 1x PBS-T, the secondary antibody, 1/10’000 diluted goat anti-rabbit IgG H&L (HRP) (ab97051; Abcam, Cambridge, UK) was added and the membrane was incubated at RT for 1 h. The membrane was then washed four times with 1x PB-T. To visualise the protein, Immobilon Crescendo Western HRP substrate (Merck, Kenilworth, NJ, USA) was added to the membrane. Chemiluminescence was captured with the Stella system (model 3200; Matlab Group Companies, Natick, MA, USA).

Targeted integration of the 5.3 kb HIV-1-based, dual-fluorophore vector LTatCL[M] into BACH2 and AAVS1 via CRISPR/Cas9-mediated homology directed repair

To investigate specific effects of HIV-1 integration into BACH2, four constructs were generated containing the vector LTatCL[M] flanked by BACH2 homologous arms (Fig. 1A) and integrated in Jurkat T-cells by means of the CRISPR/Cas9 technology. The constructs were integrated into intron 5 of BACH2 in (1) the in vivo observed same (s) and (2) the convergent (c) transcriptional orientation to investigate the potential impact of transcriptional orientation on HIV-1 promoter activity. Furthermore, LTatCL[M] was integrated into intron 2 of BACH2 in (3) the same and (4) the convergent transcriptional orientation to investigate the position effect of HIV-1 integration into BACH2. Intron 2 was chosen as it has not yet been observed in HIV-1 infected patients, however, it was frequently observed as HIV-1 integration locus in human CD34⁺ haematopoietic stem cells infected in vitro with HIV-1(Maldarelli et al., 2014). In addition, LTatCL[M] was integrated into intron 1 of PPP1R12C (protein phosphatase 1 regulatory subunit 12C) in again both transcriptional orientations. This genomic locus was previously identified as preferred site for integration of adeno-associated virus (AAV) DNA and designated AAVS1 (AAV integration site) (Kotin, Linden & Berns, 1992). AAVS1 is widely used as genomic safe harbour (GSH) althought not without some limitations particularly in the context of future use in gene therapy trials (Papapetrou & Schambach, 2016; Sadelain, Papapetrou & Bushman, 2011). Nevertheless, for research purposes it is considered a GSH and we expect that the HIV-1 promoter will not be silenced when integrated in this GSH. These six vector constructs and subsequently generated Jurkat T-cell clones were named BACH2_i5s, BACH2_i5c, BACH2_i2s, BACH2_i2c, AAVS1_c, and AAVS1_s (Figs. 1A and 1B).

Jurkat T-cells were nucleofected with linearised plasmids of the six LTatCL[M] constructs. Nine days post transfection, the frequencies of stably transfected Cerulean⁺/mCherry⁺ and single mCherry⁺ cells were between 0.1% and 1.8% (Fig. 1C). Cells were sorted to enrich Cerulean⁺/mCherry⁺ (i.e., active HIV-1 promoter) and single mCherry⁺ (i.e., inactive HIV-1 promoter) cells (Fig. 1D).

To obtain monoclonal cell lines, a sequential flow cytometric sorting strategy was employed. For all targeted integration variants, Cerulean⁺/mCherry⁺ and single mCherry⁺ cells were first sorted at 20 cells per well in 96-well plates and expanded in culture up to 50 days post transfection. We analysed by means of flow cytometry a total of 92 cell populations, and selected 29 for further expansion. These cells were single-cell sorted to obtain monoclonal cell lines and expanded in culture for at least 25 days prior to further characterisation. In total, 43 single cells expanded resulting in 43 monoclonal cell lines. For each targeted integration variant (BACH2_i5s, BACH2_i5c, BACH2_i2s, BACH2_i2c, AAVS1_s and AAVS1_c), at least one monoclonal cell line for each of the two phenotypes (Cerulean⁺/mCherry⁺ and single mCherry⁺) were obtained with the exception of AAVS1_c, single mCherry⁺ (Table 1). The monoclonal cell lines were characterised as follows.

Table 1:

Characteristics of monoclonal Jurkat T-cell lines after targeted integration of LTatCL[M] into BACH2 and AAVS1.

Cerulean⁺/mCherry⁺ and single mCherry⁺ representing active and inactive HIV-1 promoters, respectively, in LTatCL[M] transfected cells. The in vivo observed preferential HIV-1 integration loci in BACH2, BACH2_i5s, is highlighted by a red box.

LTatCL[M] integration site and orientation	HIV-1 promoter phenotype1	Monoclonal cell lines (n)	Targeted integration confirmed by PCR (%)	Mono-allelic targeted integration (%)	Unaltered BACH2 mRNA expression (%)	Unaltered BACH2 protein expression (%)
	Active	6	5/6 (83.3)	5/5 (100)	2/2 (100)	5/5 (100)
	Inactive	7	5/7 (71.4)	5/5 (100)	n.a.	4/4 (100)
BACH2_i5c	Active	4	4/4 (100)	4/4 (100)	2/2 (100)	4/4 (100)
	Inactive	1	1/1 (100)	1/1 (100)	n.a.	1/1 (100)
BACH2_i2s	Active	7	7/7 (100)	7/7 (100)	2/2 (100)	7/7 (100)
	Inactive	4	3/4 (75)	3/3 (100)	n.a.	3/3 (100)
BACH2_i2c	Active	4	4/4 (100)	4/4 (100)	2/2 (100)	4/4 (100)
	Inactive	2	2/2 (100)	2/2 (100)	n.a.	2/2 (100)
AAVS1_s	Active	2	2/2 (100)	2/2 (100)	1/1 (100)	2/2 (100)
	Inactive	3	3/3 (100)	3/3 (100)	n.a.	n.a.
AAVS1_c	Active	3	3/3 (100)	3/3 (100)	2/2 (100)	3/3 (100)
	Inactive	n.a.	n.a.	n.a.	n.a.	n.a.
Total		43	39/43 (90.7)	39/39 (100)	11/11 (100)	35/35 (100)

DOI: 10.7717/peerj.10321/table-1

Notes:

1 Active= Cerulean⁺/mCherry⁺, inactive = single mCherry⁺

n.a. = not available

First, targeted integration of the HIV-1-based, dual-fluorophore vector LTatCL[M] was verified by amplification of a fragment spanning the targeted gene up- or downstream of the 5′ or 3′ homologous arms, respectively, and LTatCL[M]. Targeted integration was confirmed in 39/43 monoclonal cell lines representing all three genomic loci in both orientations and for both fluorescence phenotypes, except for AAVS1_c single mCherry⁺ (Table 1).

Second, targeted integration of LTatCL[M] in those 39 monoclonal cell lines was verified to be mono-allelic by a PCR strategy that allows sufficient amplification only of the not targeted BACH2 or AAVS1 allele by using primers up- and downstream of the BACH2 or AAVS1 homologeous arms (Fig. 1A) and short PCR extension times. All monoclonal cell lines contained a not targeted allele of the respective targeted integration site (Table 1).

Third, BACH2 mRNA expression was quantified at 120 days post sorting in 11 Cerulean⁺/mCherry⁺ monolonal cell lines, representing each targeted integration variant. A decrease of BACH2 mRNA expression could not be observed in any Cerulean⁺/mCherry⁺ monolonal cell line (Fig. 2A; Table 1).

Forth, the potential impact on BACH2 protein expression after targeted integration of LTatCL[M] into BACH2 was investigated by Western blot analyses at 120 days post sorting. In line with unaltered BACH2 mRNA expression, BACH2 protein expression was not impaired compared to non-transfected Jurkat T-cells in all 35 monoclonal cell lines tested (Fig. 2B; Table 1).

Fifth, HIV-1 integration can affect various aspects of cellular physiology, for instance, cell proliferation. To test whether targeted integration of LTatCL[M] into BACH2 would lead to an evolutionary advantage in cell growth, a cell-growth competition assay was performed. The stably Cerulean and mCherry expressing BACH2_i5s_1.1 Cerulean⁺/mCherry⁺ and AAVS1_s_2.1 Cerulean⁺/mCherry⁺ monoclonal cell lines were combined in an approximately 1:1 ratio with the parental Jurkat T-cell line and cocultured for 25 days. A comparable decrease of Cerulean⁺/mCherry⁺ expression was observed for both the BACH2_i5s_1.1 Cerulean⁺/mCherry⁺ and the AAVS1_s_2.1 Cerulean⁺/mCherry⁺ monoclonal cell line (Fig. S5).

In summary, we successfully performed targeted integration of the 5.3 kb HIV-1-based, dual-fluorophore vector LTatCL[M] into various loci of the human genome via CRISPR/Cas9-mediated homology-directed repair. We subsequently generated monoclonal cell lines modelling actively and latently HIV-1-infected cells with integrated LTatCL[M] in either transcriptional orientation in the targeted genomic loci.

Silencing of the HIV-1 promoter in Jurkat T-cells with integrated LTatCL[M] in BACH2

To study the HIV-1 promoter activity over time in Cerulean⁺/mCherry⁺ monoclonal cell lines, that is active HIV-1 promoter, the fluorescence profile was frequently monitored for 162 days. In the majority of Cerulean⁺/mCherry⁺ monoclonal cell lines with integrated LTatCL[M] in BACH2, the HIV-1 promoter was gradually silenced as observed by the decline of the frequency of Cerulean⁺/mCherry⁺ cells to <50% in 16/18 monoclonal cell lines within 24–162 days and to <10% in 10/18 monoclonal cell lines within 58–162 days (Fig. 3A; Fig. S1). This was observed independent of the BACH2 introns 2 or 5 chosen for targeted integration of LTatCL[M] and the transcriptional orientation of LTatCL[M] relative to BACH2. In contrast, in all 5 AAVS1_s and AAVS1_c Cerulean⁺/mCherry⁺ monoclonal cell lines the frequency of Cerulean⁺/mCherry⁺ cells remained relatively stable for 162 days (>80% in 4/5 cell clones) as observed for one BACH2_i5s clone (Fig. 3A). Overall, the HIV-1 promoter when integrated into the BACH2 gene is silenced over time in the majority of Cerulean⁺/mCherry⁺ monoclonal cell lines whereas it remains active when integrated into AAVS1.

Silenced HIV-1 promoters can be reactivated by TNF-α and Romidepsin

To evaluate the reactivation of silenced HIV-1 promoters in Cerulean⁺/mCherry⁺ monoclonal cell lines, which gradually lost Cerulean expression, we performed an activation assay using TNF-α and Romidepsin. TNF-α and Romidepsin in combination have been shown to activate the silenced HIV-1 promoter (Kok et al., 2018; Sogaard et al., 2015). These compounds were used to evaluate the reactivability of the silenced HIV-1 promoter in 8 Cerulean⁺/mCherry⁺ monoclonal cell lines with integrated LTatCL[M] in BACH2. After 99 days, 1.6–48.2% of cells were Cerulean⁺/mCherry⁺. Upon treatment with TNF-α and Romidepsin, the frequencies of Cerulean⁺/mCherry⁺ cells increased to 61.6–89.8% (Fig. 3B), demonstrating that the silenced HIV-1 promoter was reactivatable in those monoclonal cell lines.

Monoclonal single mCherry⁺ cell lines harbour large internal deletions in LTatCL[M]

A total of 17 single mCherry⁺ monoclonal cell lines, presumably representing latently HIV-1-infected cells, silenced at an early time point upon targeted HIV-1 integration, were treated with TNF-α and Romidepsin to activate the HIV-1 promoter. Cerulean expression was not induced by TNF-α and Romidepsin in any of these 17 monoclonal cell lines (Fig. S1). To further investigate this, the whole vector LTatCL[M] was amplified and sequenced in 8 of these 17 monoclonal cell lines. Surprisingly, the integrated vector LTatCL[M] in these monoclonal single mCherry⁺ cell lines harboured large internal deletions in the 5’HIV-1 LTR, tat and/or Cerulean cassette (Fig. 4). The monoclonal BACH2_i5s cell lines 3.1 and 3.2, which were derived from the same cell culture after the first sort, showed the same deletion in the IRES-Cerulean region (Fig. 4A). A similar observation was made in the monoclonal BACH2_i2c cell lines 13.1 and 13.3, also derived from the same first sorted cell population: The same deletion spanning from the 5′LTR to the Cerulean cassette was observed in both monoclonal cell lines (Fig. 4B). All independent single mCherry⁺ monoclonal cell lines contained large deletions in different regions of the vector suggesting that the plasmids were not the source of those deletions. This was confirmed by next-generation sequencing of the plasmids not showing any evidence for large deletions. Next, the integrated vector LTatCL[M] was sequenced in a cell population of single mCherry⁺ cells immediately after the first sort, 9 days post transfection (Fig. S1). Evidence for numerous different large deletions were observed in the 5’HIV-1 LTR, Tat, IRES and/or Cerulean (Fig. S1), indicating that those deletions occurred early during targeted integration.