Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9.

Crispr/cas9 ablation of integrated hiv-1 accumulates proviral dna circles with reformed long terminal repeats

Maggi F.;
2021-01-01

Abstract

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9.
CRISPR/Cas9; Endonuclease; Endonucleases; Gene editing; Gene therapy; HIV-1; J-Lat; Latent infection; Latent reservoir; Rev; Tat; CRISPR-Associated Protein 9; Gene Editing; Gene Expression Regulation, Viral; Genetic Therapy; HEK293 Cells; HIV Infections; HIV-1; Humans; Proviruses; RNA, Guide; CRISPR-Cas Systems; DNA, Circular; Terminal Repeat Sequences
Lai, M.; Maori, E.; Quaranta, P.; Matteoli, G.; Maggi, F.; Sgarbanti, M.; Crucitta, S.; Pacini, S.; Turriziani, O.; Antonelli, G.; Heeney, J. L.; Freer, G.; Pistello, M.
File in questo prodotto:
File Dimensione Formato  
Lai et al. jvi.01358-21.pdf

accesso aperto

Tipologia: Versione Editoriale (PDF)
Licenza: Creative commons
Dimensione 5.61 MB
Formato Adobe PDF
5.61 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11383/2123550
Citazioni
  • ???jsp.display-item.citation.pmc??? 0
  • Scopus 8
  • ???jsp.display-item.citation.isi??? 8
social impact