Programmable Inhibition and Detection of RNA Viruses Using Cas13

Molecular Cell, Journal Year: 2019, Volume and Issue: 76(5), P. 826 - 837.e11

Published: Oct. 10, 2019

Highlights•Thousands of potential Cas13 target sites are present in ssRNA viral genomes•Cas13 is a potent antiviral against three diverse viruses cell culture•Optimal and design criteria identified via genome-wide screen•Companion Cas13-based diagnostics can be used to assess the effects targetingSummaryThe CRISPR effector could an effective for single-stranded RNA (ssRNA) because it programmably cleaves RNAs complementary its (crRNA). Here, we computationally identify thousands crRNA hundreds species that potentially infect humans. We experimentally demonstrate Cas13's activity distinct viruses: lymphocytic choriomeningitis virus (LCMV); influenza A (IAV); vesicular stomatitis (VSV). Combining this with diagnostics, develop Cas13-assisted restriction expression readout (CARVER), end-to-end platform uses detect destroy RNA. further screen crRNAs along LCMV genome evaluate how conservation nucleotide content influence activity. Our results harnessed wide range CARVER's broad utility rapid diagnostic drug development.Graphical abstract

Language: Английский

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Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing

Biologics, Journal Year: 2021, Volume and Issue: Volume 15, P. 353 - 361

Published: Aug. 1, 2021

Abstract: Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, accurate method of genome editing tool in all living cells utilized many applied disciplines. Guide RNA (gRNA) CRISPR-associated proteins are two essential components CRISPR/Cas-9 system. The mechanism contains three steps, recognition, cleavage, repair. designed sgRNA recognizes target sequence gene interest through a complementary base pair. While Cas-9 nuclease makes double-stranded breaks at site 3 pair upstream to protospacer adjacent motif, then break repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. genome-editing has wide number applications areas including medicine, agriculture, biotechnology. In it could help design new grains improve nutritional value. being investigated for cancers, HIV, therapy such as sickle cell disease, cystic fibrosis, Duchenne muscular dystrophy. technology also regulation specific genes advanced modification protein. However, immunogenicity, effective delivery systems, off-target effect, ethical issues have been major barriers extend clinical applications. Although becomes era molecular biology countless roles ranging from basic researches applications, there still challenges rub practical various improvements needed overcome obstacles. Keywords: CRISPR, Cas-9, sgRNA, gene-editing, mechanism,

Language: Английский

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Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice

Nature Communications, Journal Year: 2019, Volume and Issue: 10(1)

Published: July 2, 2019

Abstract Elimination of HIV-1 requires clearance and removal integrated proviral DNA from infected cells tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) CRISPR-Cas9 demonstrate viral in latent infectious reservoirs humanized mice. subgenomic fragments, spanning the long terminal repeats Gag gene, are excised vivo, resulting elimination DNA; virus is not detected blood, lymphoid tissue, bone marrow brain by nested digital-droplet PCR as well RNAscope tests. No mediated off-target effects detected. Adoptive transfer human immunocytes dual treated, virus-free animals to uninfected mice fails produce progeny virus. In contrast, readily following sole LASER ART or treatment. These data provide proof-of-concept that permanent possible.

Language: Английский

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Application of CRISPR/Cas9-Based Gene Editing in HIV-1/AIDS Therapy

Frontiers in Cellular and Infection Microbiology, Journal Year: 2019, Volume and Issue: 9

Published: March 22, 2019

Despite the fact that great efforts have been made in prevention and resistance of HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. Highly active antiretroviral therapy (HAART) can suppress virus replication, but it cannot eradicate latent viral reservoirs patients. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has engineered as an effective gene-editing technology with potential treat HIV-1/AIDS. It be used target cellular co-factors or genome reduce infection clear provirus, well induce transcriptional activation for elimination. This versatile gene editing successfully applied reduction cells animal models. Here, we update rapid progress CRISPR/Cas9-based research recent years discuss limitations future perspectives its application.

Language: Английский

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CRISPR/Cas gene therapy

Journal of Cellular Physiology, Journal Year: 2020, Volume and Issue: 236(4), P. 2459 - 2481

Published: Sept. 22, 2020

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated enzyme (Cas) is a naturally occurring genome editing tool adopted from the prokaryotic adaptive immune defense system. Currently, CRISPR/Cas9‐based has been becoming one of most promising tools for treating human genetic diseases, including cardiovascular neuro‐disorders, and cancers. As quick modification CRISPR/Cas9 system, delivery gene therapy extensively studied in preclinic clinic treatments. CRISPR/Cas also robust to create animal models studying disorders, particularly diseases associated with point mutations. However, significant challenges remain before technology can be routinely employed different which include toxicity response treated cells component, highly throughput method, potential off‐target impact. The effect major concerns therapy, more research should focused on limiting this impact by designing high specific gRNAs using specificity Cas enzymes. Modifying method not only targets tissue/cell but potentially limits

Language: Английский

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

Proceedings of the National Academy of Sciences, Journal Year: 2023, Volume and Issue: 120(19)

Published: May 1, 2023

Treatment of HIV-1 ADA -infected CD34+ NSG-humanized mice with long-acting ester prodrugs cabotegravir, lamivudine, and abacavir in combination native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) proviral DNA gene editing. This led to sequential viral suppression, restoration absolute human CD4 + T cell numbers, then elimination replication-competent virus 58% infected mice. Dual CRISPR therapies enabled the excision integrated cells contained within live animals. Highly sensitive nucleic acid nested droplet digital PCR, RNAscope, outgrowth assays affirmed elimination. not detected blood, spleen, lung, kidney, liver, gut, bone marrow, brain virus-free Progeny from adoptively transferred CRISPR-treated neither nor recovered. Residual fragments were easily seen untreated viral-rebounded No evidence off-target toxicities recorded any treated Importantly, therapy demonstrated statistically significant improvements cure percentages compared single treatments. Taken together, these observations underscore a pivotal role combinatorial editing achieving infection.

Language: Английский

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Target product profile for cell-based and gene-based therapies to achieve a cure for HIV

The Lancet HIV, Journal Year: 2025, Volume and Issue: 12(2), P. e154 - e162

Published: Jan. 5, 2025

Language: Английский

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In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

F1000Research, Journal Year: 2017, Volume and Issue: 6, P. 2153 - 2153

Published: Dec. 20, 2017

Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in wide range of animal models and human clinical trials. With the advent clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one most suitable viral vectors to package, deliver, express CRISPR components for targeted gene editing. Recent discoveries smaller Cas9 orthologues have enabled packaging nuclease its chimeric guide RNA into single delivery vehicle robust vivo genome Here, we discuss how combined use small orthologues, tissue-specific minimal promoters, serotypes, different routes administration advanced development efficient precise editing comprehensively review various AAV-CRISPR systems that been effectively used animals. We then implications potential strategies overcome off-target effects, immunogenicity, toxicity associated vehicles. Finally, ongoing non-viral-based ex therapy trials underscore current challenges future prospects CRISPR/Cas9 therapeutics.

Language: Английский

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CRISPR-Cas Biology and Its Application to Infectious Diseases

Journal of Clinical Microbiology, Journal Year: 2019, Volume and Issue: 57(4)

Published: March 27, 2019

Infectious diseases remain a global threat contributing to excess morbidity and death annually, with the persistent potential for destabilizing pandemics. Improved understanding of pathogenesis bacteria, viruses, fungi, parasites, along rapid diagnosis treatment human infections, is essential improving infectious disease outcomes worldwide.

Language: Английский

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Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing

Nanoscale, Journal Year: 2017, Volume and Issue: 10(3), P. 1063 - 1071

Published: Dec. 18, 2017

GO-PEG-PEI nanocarrier, which was constructed for the delivery of high-molecular-weight Cas9/sgRNA complexes executing endocytosis, endosomal escape, nuclear entry, and gene editing.

Language: Английский

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