Abstract
Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the stable expression of CRISPR-Cas9 or Cas12a systems targeting HIV DNA. Because lentiviral transduction and long-term CRISPR-Cas activity are less suitable for in vivo application of this antiviral strategy, we investigated whether HIV can also be completely inactivated by transient CRISPR-Cas activity. Latently infected SupT1 T-cells were repeatedly transfected with different Cas9 and Cas12a mRNA/protein sources in combination with dual gRNAs/crRNAs targeting highly conserved viral sequences. Upon repeated Cas9 protein treatment, viral replication could no longer be reactivated. We demonstrate that this was due to complete mutational inactivation of the proviral DNA, mostly through mutations at the target sites, but also through excision or inversion of the viral DNA fragment between the two target sites. These results demonstrate that repeated transient CRISPR-Cas treatment of a latently infected T-cell culture can lead to the permanent inactivation of HIV replication, indicating that transient CRISPR-Cas delivery methods can be considered for in vivo application.
Highlights
When mammalian cells were transfected with different CRISPR-Cas9 formats for genome editing, Cas9 protein was found to be more efficient than Cas9 mRNA [35,36]
The higher efficiency of the Cas9 protein is largely due to the fact that the protein was transfected into the nucleus as a pre-assembled Cas9 protein-guide RNA (gRNA) ribonucleoprotein (RNP) complex that can immediately cleave the target DNA, whereas upon transfection of the mRNA, the mRNA first needs to be translated in the cytoplasm and the protein product has to complex with the gRNA and enter the nucleus before the DNA can be cleaved
Upon microinjection of Cas9 or Cas12a protein combined with different gRNAs/CRISPR RNA (crRNA) into zebrafish embryos, both Cas proteins resulted in high overall gene editing, but the editing efficiency measured with the Cas12a protein varied substantially for different crRNAs and was relatively low for several crRNAs [37]
Summary
Since the discovery of HIV as the causative agent for AIDS almost 40 years ago, extensive research has resulted in antiretroviral therapy (ART) with a combination of drugs that can reduce the viral load in HIV-infected patients to undetectable levels, but does not permanently cure the patient. These drugs target essential processes in the viral replication cycle, such as reverse transcription of the viral RNA genome into a DNA copy and integration of this DNA in the cellular genome, but do not inactivate the proviral DNA in infected reservoir cells [1,2,3].
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