scholarly journals Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects

2021 ◽  
Vol 12 ◽  
Author(s):  
Yufan Xu ◽  
Xiaorong Peng ◽  
Yanghao Zheng ◽  
Changzhong Jin ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Lentiviral Vector ◽  
Host Cells ◽  
Preliminary Evaluation ◽  
Major Barrier ◽  
Editing Efficiency ◽  
Latent Hiv ◽  
Hiv 1 ◽  
Target Effects

Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology.

Resveratrol Promotes HIV-1 Tat Accumulation via AKT/FOXO1 Signaling Axis and Potentiates Vorinostat to antagonize HIV-1 latency

2021 ◽  
Vol 19 ◽  
Author(s):  
Zeming Feng ◽  
Zhengrong Yang ◽  
Xiang Gao ◽  
Yuhua Xue ◽  
Xiaohui Wang
Keyword(s):  
Host Cells ◽  
Latent Reservoir ◽  
Cell Models ◽  
Elongation Complex ◽  
Major Barrier ◽  
Small Nuclear Ribonucleoprotein ◽  
Candidate Drug ◽  
Signaling Axis ◽  
Shock And Kill ◽  
Hiv 1

Background: The latent reservoir of HIV-1 is a major barrier to achieving the eradication of HIV-1/AIDS. One strategy is termed “shock and kill”, which aims to awaken the latent HIV-1 using latency reversing agents (LRAs) to replicate and produce HIV-1 particles. Subsequently, the host cells containing HIV-1 can be recognized and eliminated by the immune response and anti-retroviral therapy. Although many LRAs have been found and tested, their clinical trials were dissatisfactory. Objective: To study how resveratrol reactivating silent HIV-1 transcription and assess if resveratrol could be a candidate drug for the “shock” phase in “shock and kill” strategy. Method: We used established HIV-1 transcription cell models (HeLa-based NH1 and NH2 cells) and HIV-1 latent cell models (J-Lat A72 and Jurkat 2D10 cells). We performed resveratrol treatment on these cell lines and studied the mechanism of how resveratrol stimulating HIV-1 gene transcription. We also tested resveratrol’s bioactivity on primary cells isolated from HIV1 latent infected patients. Results: Resveratrol promoted HIV-1 Tat protein levels, and resveratrol-induced Tat promotion was dependent on the AKT/FOXO1 signaling axis. Resveratrol could partially dissociate P-TEFb (Positive Transcription Elongation Factor b) from 7SK snRNP (7SK small nuclear Ribonucleoprotein) and promote Tat-SEC (Super Elongation Complex) interaction. Preclinical studies showed that resveratrol potentiated Vorinostat to awaken HIV-1 latency in HIV-1 latent infected cells isolated from patients. Conclusion: We found a new mechanism of resveratrol stimulating the production of HIV-1. Resveratrol could be a promising candidate drug to eradicate HIV-1 reservoirs.

scholarly journals Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR

2021 ◽  
Author(s):  
Gregory A. Viglianti ◽  
Vicente Planelles ◽  
Timothy M. Hanley
Keyword(s):  
Adaptor Protein ◽  
Type I ◽  
Major Barrier ◽  
E Coli ◽  
Tissue Microenvironment ◽  
Viral Spread ◽  
Latent Hiv ◽  
Hiv 1

Macrophages are infected by HIV-1 in vivo and contribute to both viral spread and pathogenesis. Recent human and animal studies suggest that HIV-1-infected macrophages serve as a reservoir that contributes to HIV-1 persistence during anti-retroviral therapy. The ability of macrophages to serve as persistent viral reservoirs is likely influenced by the local tissue microenvironment, including interactions with pathogenic and commensal microbes. Here we show that the sexually transmitted pathogen Neisseria gonorrhoeae (GC) and the gut-associated microbe Escherichia coli (E. coli), which encode ligands for both Toll-like receptor 2 (TLR2) and TLR4, repressed HIV-1 replication in macrophages and thereby induced a state reminiscent of viral latency. This repression was mediated by signaling through TLR4 and the adaptor protein TRIF and was associated with increased production of type I interferons. Inhibiting TLR4 signaling, blocking type 1 interferon, or knocking-down TRIF reversed LPS- and GC-mediated repression of HIV-1. Finally, the repression of HIV-1 in macrophages was associated with the recruitment of interferon regulatory factor 8 (IRF8) to the interferon stimulated response element (ISRE) downstream of the 5’ HIV-1 long terminal repeat (LTR). Our data indicate that IRF8 is responsible for repression of HIV-1 replication in macrophages in response to TRIF-dependent signaling during GC and E. coli co-infection. These findings highlight the potential role of macrophages as HIV-1 reservoirs as well as the role of the tissue microenvironment and co-infections as modulators of HIV-1 persistence. IMPORTANCE The major barrier toward the eradication of HIV-1 infection is the presence of a small reservoir of latently infected cells, which include CD4+ T cells and macrophages that escape immune-mediated clearance and the effects of anti-retroviral therapy. There remain crucial gaps in our understanding of the molecular mechanisms that lead to transcriptionally silent or latent HIV-1 infection of macrophages. The significance of our research is in identifying microenvironmental factors, such as commensal and pathogenic microbes, that can contribute to the establishment and maintenance of latent HIV-1 infection in macrophages. It is hoped that identifying key processes contributing to HIV-1 persistence in macrophages may ultimately lead to novel therapeutics to eliminate latent HIV-1 reservoirs in vivo.

scholarly journals Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

2019 ◽  
Vol 20 (15) ◽  
pp. 3719 ◽  
Author(s):  
Zahra Hajiahmadi ◽  
Ali Movahedi ◽  
Hui Wei ◽  
Dawei Li ◽  
Yasin Orooji ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Short Palindromic Repeat ◽  
External Forces ◽  
Time And Energy ◽  
Reduced Time ◽  
Target Effects

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3006-3015 ◽  
Author(s):  
Joseph Kulkosky ◽  
Derek M. Culnan ◽  
Jeanette Roman ◽  
Geethanjali Dornadula ◽  
Matthias Schnell ◽  
...  
Keyword(s):  
Adjuvant Therapy ◽  
Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Receptor Expression ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Down Regulation ◽  
Viral Spread ◽  
Latent Hiv ◽  
Hiv 1

Abstract Prostratin is a unique phorbol ester that stimulates protein kinase C activity but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo human immunodeficiency virus type 1 (HIV-1) infection yet up-regulate viral expression from latent proviruses. Prostratin's lack of tumor promotion, coupled with its ability to block viral spread yet induce latent proviral expression, prompted studies to determine whether this compound could serve as an inductive adjuvant therapy for patients treated with highly active antiretroviral therapy (HAART). The current experiments indicate that prostratin is a potent mitogen for mononuclear phagocytes possessing many of the activities of phorbol myristate acetate (PMA) with notable functional differences. Prostratin, like PMA, accelerates differentiation of the myeloid cell-lines, HL-60 and THP-1, as well as mononuclear phagocytes from bone marrow and peripheral blood. Enzyme-linked immunosorbent assay and gene array analyses indicate significant changes in the expression of proteins and messenger RNA after treatment of cells with prostratin, consistent with phagocyte activation and differentiation. Prostratin blocks HIV-1 infection relating to down-regulation of CD4 receptor expression. The array analysis indicates a similar down-regulation of the HIV-1 coreceptors, CXCR4 and CCR5, and this may also reduce viral infectivity of treated host cells. Finally, prostratin is capable of up-regulating HIV-1 expression from CD8+ T lymphocyte–depleted peripheral blood mononuclear cells of patients undergoing HAART. This novel observation suggests the agent may be an excellent candidate to augment HAART by inducing expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in certain individuals infected with HIV-1.

scholarly journals Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice

2019 ◽  
Author(s):  
Zhuqing Wang ◽  
Yue Wang ◽  
Shawn Wang ◽  
Andrew J Gorzalski ◽  
Hayden McSwiggin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Editing Efficiency ◽  
Protospacer Adjacent Motif ◽  
Summary Statement ◽  
Strict Requirement ◽  
Moraxella Bovoculi ◽  
Target Effects

AbstractDespite many advantages over Cas9, Cas12a has not been widely used in genome editing in mammalian cells largely due to its strict requirement of the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) could edit the genome in murine zygotes independent of TTTV PAM sequences and with minimal on-target mutations and close to 100% editing efficiency when crRNAs of 23nt spacers were used.Summary statementCRISPR-Mb3Cas12a can target a broader range of sequences in murine zygotes compared to AsCas12a and LbCas12a, and has lower on-target effects than Cas9 and high overall knock-in efficiency.

scholarly journals Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

2019 ◽  
Author(s):  
Jacob Lamberth ◽  
Laura Daley ◽  
Pachai Natarajan ◽  
Stanislav Khoruzhenko ◽  
Nurit Becker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Genome Editing ◽  
Plasmid Dna ◽  
High Efficiency ◽  
High Specificity ◽  
Human Cells ◽  
Dna And Rna ◽  
Genome Modification ◽  
Delivery Technology ◽  
Target Effects

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

scholarly journals Development of an Avian Leukosis-Sarcoma Virus Subgroup A Pseudotyped Lentiviral Vector

2001 ◽  
Vol 75 (19) ◽  
pp. 9339-9344 ◽  
Author(s):  
Brian C. Lewis ◽  
Nachimuthu Chinnasamy ◽  
Richard A. Morgan ◽  
Harold E. Varmus
Keyword(s):  
Transgenic Mice ◽  
Lentiviral Vector ◽  
Transgenic Animals ◽  
Cell Types ◽  
Host Cells ◽  
Target Cells ◽  
Mouse Tumor ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACT We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 × 103 infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1 pol gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 105 IU/ml and can be concentrated by ultracentrifugation to titers of 107 IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the β-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.

scholarly journals CRISPR/Cas9 Genome Editing to Disable the Latent HIV-1 Provirus

2018 ◽  
Vol 9 ◽  
Author(s):  
Amanda R. Panfil ◽  
James A. London ◽  
Patrick L. Green ◽  
Kristine E. Yoder
Keyword(s):  
Genome Editing ◽  
Latent Hiv ◽  
Hiv 1

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

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1154
Author(s):  
Alex Olson ◽  
Binita Basukala ◽  
Seunghee Lee ◽  
Matthew Gagne ◽  
Wilson W. Wong ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Major Barrier ◽  
Guide Rnas ◽  
Immunodeficiency Virus ◽  
Shock And Kill ◽  
Infected Cells ◽  
Latent Hiv ◽  
Transcriptional Repressor Domain ◽  
Hiv 1 ◽  
Latent Provirus

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.

scholarly journals Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Soo-Ji Park ◽  
Tae Yeong Jeong ◽  
Seung Kyun Shin ◽  
Da Eun Yoon ◽  
Soo-Yeon Lim ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Genome Editing ◽  
Mouse Models ◽  
Targeted Mutagenesis ◽  
Mutant Mice ◽  
Germline Transmission ◽  
Dwarf Phenotype ◽  
Editing Efficiency ◽  
Mouse Cells ◽  
Target Effects

AbstractPrime editors, novel genome-editing tools consisting of a CRISPR-Cas9 nickase and an engineered reverse transcriptase, can induce targeted mutagenesis. Nevertheless, much effort is required to optimize and improve the efficiency of prime-editing. Herein, we introduce two strategies to improve the editing efficiency using proximal dead sgRNA and chromatin-modulating peptides. We used enhanced prime-editing to generate Igf2 mutant mice with editing frequencies of up to 47% and observed germline transmission, no off-target effects, and a dwarf phenotype. This improved prime-editing method can be efficiently applied to cell research and to generate mouse models.

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