Regulation of Cas9 by viral proteins Tat and Rev for HIV-1 inactivation.

While combined antiretroviral therapy (cART) has had a great impact on the treatment of HIV-1 infection, the persistence of long-lived cells with an intact provirus precludes virus eradication and sterilizing cure. CRISPR/Cas9 genome editing has become an efficient tool to eradicate HIV-1 genome or prevent replication. Furthermore, regulation of Cas9 gene expression by HIV can induce mutations that could inactivate the proviral genome, making a gene therapy safe by preventing the induction of non-specific mutations, which could compromise the integrity of healthy cells. In this study, isolated HIV-1 LTR, INS and RRE sequences were used to regulate Cas9 expression in HEK293 cells, and guide RNAs (gRNAs) were designed to target mutations in HIV-1 conserved regions such as tat and rev regulatory genes. We demonstrate that Cas9 expression in our system is controlled by the HIV-1 Tat and Rev proteins, leading to self-regulation of gene edition, and showing a strong antiviral effect by inactivating HIV-1 replication. Sequencing analysis confirmed that viral genome was partially excised by multiplex editing (90% efficiency), and viral capsid protein (CA-p24) was undetectable. In conclusion, the self-regulated CRISPR/Cas9 system may be a reliable and accurate strategy for eliminating HIV-1 infection whose effect will be restricted to infected cells.

Vergara-Mendoza M ，Gomez-Quiroz LE ，Miranda-Labra RU ，Fuentes-Romero LL ，Romero-Rodríguez DP ，González-Ruiz J ，Hernández-Rizo S ，Viveros-Rogel M ... -  《-》

CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats.

Lai M ，Maori E ，Quaranta P ，Matteoli G ，Maggi F ，Sgarbanti M ，Crucitta S ，Pacini S ，Turriziani O ，Antonelli G ，Heeney JL ，Freer G ，Pistello M ... -  《-》

Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape.

Ophinni Y ，Miki S ，Hayashi Y ，Kameoka M ... -  《Viruses-Basel》

CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination.

A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery. A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels. Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing. These results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing. The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.

Herskovitz J ，Hasan M ，Patel M ，Blomberg WR ，Cohen JD ，Machhi J ，Shahjin F ，Mosley RL ，McMillan J ，Kevadiya BD ，Gendelman HE ... -  《EBioMedicine》

Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA.

Smith LM ，Ladner JT ，Hodara VL ，Parodi LM ，Harris RA ，Callery JE ，Lai Z ，Zou Y ，Raveedran M ，Rogers J ，Giavedoni LD ... -  《-》