Silencing integrated SIV proviral DNA with TAR-specific CRISPR tools.

Area of Science:

• Virology
• Molecular Biology
• Gene Editing

Background:

• A functional cure for HIV requires preventing transcription from integrated proviral DNA.
• The Tat protein's interaction with the TAR element RNA is crucial for HIV transcription.
• This study investigates blocking the Tat-TAR interaction using the SIVmac model.

Purpose of the Study:

• To test the efficacy of blocking the Tat-TAR interaction in SIV transcription.
• To evaluate CRISPR-Cas9 technology for targeting the SIVmac TAR element.
• To explore strategies for a functional cure of HIV.

Main Methods:

• Designed five CRISPR short guiding RNAs (sgRNAs) targeting the SIVmac TAR element.
• Utilized inactive Cas9 (dCas9) and dCas9-KRAB effectors.
• Delivered sgRNA constructs as ribonucleoproteins or plasmid DNA into cells with SIV DNA or integrated viral DNA.

Main Results:

• sgRNAs targeting the TAR element's coding strand inhibited SIV RNA transcription.
• Inhibition was observed specifically when using dCas9-KRAB, not dCas9 alone.
• This suggests a role for epigenetic modification in transcriptional inhibition.

Conclusions:

• CRISPR-mediated targeting of the TAR element, particularly with epigenetic modifiers like KRAB, can inhibit SIV transcription.
• Epigenetic modifications may offer a more effective strategy for proviral inactivation than simple transcriptional interference.
• This approach holds potential for developing a functional cure for HIV in vivo.