Suppression of HSV-1 infection and viral reactivation by CRISPR-Cas9 gene editing in 2D and 3D culture models.

Although our understanding of herpes simplex virus type 1 (HSV-1) biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR-Cas9-based gene editing platform to specifically target the HSV-1 genome.

Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates.

In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.

Strategic self-limiting production of infectious HIV particles by CRISPR in permissive cells.

Post-translational glycosylation of the HIV-1 envelope protein involving precursor glycan trimming by mannosyl oligosaccharide glucosidase (MOGS) is critically important for morphogenesis of virions and viral entry. Strategic editing of the MOGS gene in T lymphocytes and myeloid origin cells harboring latent proviral DNA results in the production of non-infectious particles upon treatment of cells with latency reversal agents. Controlled activation of CRISPR-MOGS by rebound HIV-1 mitigates production of infectious particles that exhibit poor ability of the virus to penetrate uninfected cells.

CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice.

Treatment of HIV-1-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4 T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals.

Coronavirus infection in chemosensory cells.

Clinical manifestations of human coronavirus (HCoV)-related diseases are mostly related to the respiratory system, although secondary complications such as headache, anosmia, ageusia, and myalgia have been reported. HCoV infection and replication in chemosensory cells associated with ageusia and anosmia is poorly understood. Here, we characterized HCoV-OC43 and SARS-CoV-2 infection in two types of chemosensory cells, olfactory and taste cells, with their unique molecular and histological characteristics.

Polθ promotes the repair of 5'-DNA-protein crosslinks by microhomology-mediated end-joining.

DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts.

CRISPR based editing of SIV proviral DNA in ART treated non-human primates.

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues.

Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5' Adducts.

Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5' end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc).

The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair.

The key event in the choice of repair pathways for DNA double-strand breaks (DSBs) is the initial processing of ends. Non-homologous end joining (NHEJ) involves limited processing, but homology-dependent repair (HDR) requires extensive resection of the 5' strand. How cells decide if an end is channeled to resection or NHEJ is not well understood.

DNA double-strand breaks with 5' adducts are efficiently channeled to the DNA2-mediated resection pathway.

DNA double-strand breaks (DSBs) with 5' adducts are frequently formed from many nucleic acid processing enzymes, in particular DNA topoisomerase 2 (TOP2). The key intermediate of TOP2 catalysis is the covalent complex (TOP2cc), consisting of two TOP2 subunits covalently linked to the 5' ends of the nicked DNA. In cells, TOP2ccs can be trapped by cancer drugs such as etoposide and then converted into DNA double-strand breaks (DSBs) that carry adducts at the 5' end.

The N-terminus of RPA large subunit and its spatial position are important for the 5'->3' resection of DNA double-strand breaks.

The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5' strand to generate 3' ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5'->3' directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA.

Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.

The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails.

Analysis of MRE11's function in the 5'-->3' processing of DNA double-strand breaks.

The resection of DNA double-strand breaks (DSBs) into 3' single-strand tails is the initiating step of homology-dependent repair pathways. A key player in this process is the MRE11-RAD50-NBS1 complex, but its contribution to and mechanistic role in resection are not well understood. In this study, we took advantage of the Xenopus egg extract system to address these questions.

Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration.

During cotranslational integration of a eukaryotic multispanning polytopic membrane protein (PMP), its hydrophilic loops are alternately directed to opposite sides of the ER membrane. Exposure of fluorescently labeled nascent PMP to the cytosol or ER lumen was detected by collisional quenching of its fluorescence by iodide ions localized in the cytosol or lumen. PMP loop exposure to the cytosol or lumen was controlled by structural rearrangements in the ribosome, translocon, and associated proteins that occurred soon after a nascent chain transmembrane segment (TMS) entered the ribosomal tunnel.

Mechanistic analysis of Xenopus EXO1's function in 5'-strand resection at DNA double-strand breaks.

The processing of DNA double-strand breaks (DSBs) into 3' single-stranded tails is the first step of homology-dependent DSB repair. A key player in this process is the highly conserved eukaryotic exonuclease 1 (EXO1), yet its precise mechanism of action has not been rigorously determined. To address this issue, we reconstituted 5'-strand resection in cytosol derived from unfertilized interphase eggs of the frog Xenopus laevis.

Replication protein A promotes 5'-->3' end processing during homology-dependent DNA double-strand break repair.

Replication protein A (RPA), the eukaryotic single-strand deoxyribonucleic acid (DNA [ss-DNA])-binding protein, is involved in DNA replication, nucleotide damage repair, mismatch repair, and DNA damage checkpoint response, but its function in DNA double-strand break (DSB) repair is poorly understood. We investigated the function of RPA in homology-dependent DSB repair using Xenopus laevis nucleoplasmic extracts as a model system. We found that RPA is required for single-strand annealing, one of the homology-dependent DSB repair pathways.

Identification of the Xenopus DNA2 protein as a major nuclease for the 5'->3' strand-specific processing of DNA ends.

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5' strand-specific processing of DNA ends to generate 3' single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5' strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5'-->3' degradation of single-strand tails.

Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts.

Cellular DNA is under constant attack from numerous exogenous and endogenous agents. The resulting DNA lesions, if not repaired timely, could stall DNA replication, leading to genome instability. To better understand the mechanism of DNA lesion replication at the biochemical level, we have attempted to reconstitute this process in Xenopus egg extracts, the only eukaryotic in vitro system that relies solely on cellular proteins for DNA replication.