Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

Nerys-Junior, Arildo; Costa, Lendel Correia da; Braga-Dias, Luciene P; Oliveira, Márcia Pereira de; Rossi, Átila Duque; Cunha, Rodrigo Delvecchio da; Gonçalves, Gabriel Santos; Tanuri, Amílcar

doi:10.1590/s1415-47572014000100018

Cited by 6 publications

(9 citation statements)

References 20 publications

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“…However, these strategies do not silence CCR5 expression permanently. To reach this goal, some gene editing technologies, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been used to knock out the CCR5 gene [ 56 , 57 ]. Several clinical trials using autologous infusions of CCR5 -modified CD4 + T cells are currently under way.…”

Section: Full or Partial Replacement Of The Immune System Through Genmentioning

confidence: 99%

HIV-1 functional cure: will the dream come true?

Liu

et al. 2015

BMC Med

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The reservoir of human immunodeficiency virus type 1 (HIV-1), a long-lived pool of latently infected cells harboring replication-competent viruses, is the major obstacle to curing acquired immune deficiency syndrome (AIDS). Although the combination antiretroviral therapy (cART) can successfully suppress HIV-1 viremia and significantly delay the progression of the disease, it cannot eliminate the viral reservoir and the patient must continue to take anti-viral medicines for life. Currently, the appearance of the ‘Berlin patient’, the ‘Boston patients’, and the ‘Mississippi baby’ have inspired many therapeutic strategies for HIV-1 aimed at curing efforts. However, the specific eradication of viral latency and the recovery and optimization of the HIV-1-specific immune surveillance are major challenges to achieving such a cure. Here, we summarize recent studies addressing the mechanisms underlying the viral latency and define two categories of viral reservoir: ‘shallow’ and ‘deep’. We also present the current strategies and recent advances in the development of a functional cure for HIV-1, focusing on full/partial replacement of the immune system, ‘shock and kill’, and ‘permanent silencing’ approaches.

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Section: Full or Partial Replacement Of The Immune System Through Genmentioning

confidence: 99%

HIV-1 functional cure: will the dream come true?

Liu

et al. 2015

BMC Med

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“…Fluorescence measurements by flow cytometry showed that GFP + cells were most abundant in CRISPR-Cas9-transfected cells 48 h after transfection ( Figure 3 ). Whereas Miller’s TALEN transfections resulted in ~10% of RFP + /GFP + cells ( Nerys-Junior et al , 2014 ), CRISPR-Cas9 transfections resulted in 57.2% of GFP + cells ( Figure 3 ). The transfections were repeated 10 times in the same conditions and in all cases the proportion of gated cells showed no more than 2% variability.…”

Section: Resultsmentioning

confidence: 99%

“…This same approach was previously used to show that the interval with the highest proportion of RFP + /GFP + cells in TALEN+pRGS-CR transfections was 72 h after transfection ( Kim et al. , 2011 ; Nerys-Junior et al , 2014 ) and this interval was used in cell sorting of TALEN+pRGS-CR transfections. For CRISPR-Cas9-transfected HEK293T cells, the best interval (highest proportion of GFP + cells) was 48 h post-transfection.…”

Section: Methodsmentioning

confidence: 99%

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene

et al. 2018

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The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells.

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“…To detect designer nuclease induced in/del mutations, T7E1 assays or HMA 12 , 24 , 25 based on HD formation of mutated and unmutated DNA are widely used. HD based assays work independent of the size of in/dels and there is no need for special equipment.…”

Section: Discussionmentioning

confidence: 99%

Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

Ehrke‐Schulz

Bergmann

Schiwon

et al. 2016

Molecular Therapy - Methods & Clinical Development

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Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA.

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Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

Cited by 6 publications

References 20 publications

HIV-1 functional cure: will the dream come true?

HIV-1 functional cure: will the dream come true?

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene

Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

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