CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape.
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ABSTRACT:
SUBMITTER: Wang G
PROVIDER: S-EPMC4786927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach. Here, we demonstrate that CRISPR/Cas9 targeting of single HIV loci, only partially inhibits HIV replication and facilitates rapid viral escape at the target site. A combinatorial approach of two strong gRNAs targeting different regions of the HIV genome can completely abrogate viral replication and prevent viral escape. Our data shows that the accelerating effect of gene-editing on viral escape can be overcome and as such gene-editing may provide a future alternative for control of HIV-infection.
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Project description:Screening with CRISPR/Cas9 technology has already led to significant discoveries in the fields of cancer biology, cell biology and virology. Because of the relatively low false discovery rates and the ability to perform high-throughput, pooled approaches, it has rapidly become the assay of choice for screening studies, including whole-genome screens. Here, we describe a CRISPR screening protocol that allows for efficient screening of the entire life cycle of HIV-1 through packaging of the HIV-CRISPR lentiviral genomes by infecting HIV-1 virus in trans.
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Project description:Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.
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Project description:The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs.
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Project description:Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.
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Project description:Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.
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Project description:Recently the developed single guide (sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. We screened the immediate-early-1 gene (ie-1), the major envelope glycoprotein gene (gp64), and the late expression factor gene (lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector (PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.
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Project description:CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.
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Project description:The emergence of influenza A viruses (IAVs) from zoonotic reservoirs poses a great threat to human health. As seasonal vaccines are ineffective against zoonotic strains, and newly transmitted viruses can quickly acquire drug resistance, there remains a need for host-directed therapeutics against IAVs. Here, we performed a genome-scale CRISPR/Cas9 knockout screen in human lung epithelial cells with a human isolate of an avian H5N1 strain. Several genes involved in sialic acid biosynthesis and related glycosylation pathways were highly enriched post-H5N1 selection, including SLC35A1, a sialic acid transporter essential for IAV receptor expression and thus viral entry. Importantly, we have identified capicua (CIC) as a negative regulator of cell-intrinsic immunity, as loss of CIC resulted in heightened antiviral responses and restricted replication of multiple viruses. Therefore, our study demonstrates that the CRISPR/Cas9 system can be utilized for the discovery of host factors critical for the replication of intracellular pathogens.
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Project description:The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293?T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells. Tat functional assays showed significantly reduced HIV-1 promoter-driven luciferase expression after tat-CRISPR transduction, while Rev functional assays revealed abolished gp120 expression after rev-CRISPR transduction. The target gene was mutated at the Cas9 cleavage site with high frequency and various indel mutations. Conversely, no mutations were detected at off-target sites and Cas9 expression had no effect on cell viability. CRISPR/Cas9 was further tested in persistently and latently HIV-1-infected T-cell lines, in which p24 levels were significantly suppressed even after cytokine reactivation, and multiplexing all six gRNAs further increased efficiency. Thus, the CRISPR/Cas9 system targeting HIV-1 regulatory genes may serve as a favorable means to achieve functional cures.
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