Targeted Chromatinization and Repression of HIV-1 Provirus Transcription with Repurposed CRISPR/Cas9

Viruses. 2020 Oct 12;12(10):1154. doi: 10.3390/v12101154.

Abstract

The major barrier to HIV-1 cure is the persistence of latent provirus, which is not eradicated by antiretroviral therapy. The "shock and kill" approach entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. CRISPR/Cas9 has been utilized to excise integrated Human Immunodeficiency Virus (HIV) DNA from infected cells in an RNA-guided, sequence-specific manner. Here, we seek to epigenetically silence the proviral DNA by introducing nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB). We show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies.

Keywords: CRISPR; HIV latency; HIV transcription; chromatin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • CRISPR-Cas Systems
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Epigenesis, Genetic / genetics
  • HEK293 Cells
  • HIV Infections / virology
  • HIV Long Terminal Repeat / genetics
  • HIV-1 / genetics*
  • Histones / metabolism
  • Humans
  • Jurkat Cells
  • Methylation
  • Proviruses / genetics*
  • RNA, Guide, Kinetoplastida / genetics*
  • Repressor Proteins / metabolism
  • Transcription, Genetic / genetics
  • Virus Activation / genetics*
  • Virus Latency / genetics*

Substances

  • Histones
  • RNA, Guide
  • Repressor Proteins
  • ZNF350 protein, human