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The highly conserved trans-acting response element (TAR) present in the RNA genome of human immunodeficiency virus 1 (HIV-1) is a stably folded hairpin structure involved in viral replication. However, TAR is also sensed by viral sensors, leading to antiviral immunity. While high variation in the TAR RNA structure renders the virus replication-incompetent, effects on viral sensing remain unclear. Here, we investigated the role of TAR RNA structure and stability on viral sensing. TAR mutants with deletions in the TAR hairpin that enhanced thermodynamic stability increased antiviral responses. Strikingly, TAR mutants with lower stability due to destabilization of the TAR hairpin also increased antiviral responses without affecting pro-inflammatory responses. Moreover, mutations that affected the TAR RNA sequence also enhanced specific antiviral responses. Our data suggest that mutations in TAR of replication-incompetent viruses can still induce immune responses via viral sensors, hereby underscoring the robustness of HIV-1 RNA sensing mechanisms.
Melissa Stunnenberg; John van Hamme; Atze Das; Ben Berkhout; Teunis Geijtenbeek. Variations in the Abortive HIV-1 RNA Hairpin Do Not Impede Viral Sensing and Innate Immune Responses. Pathogens 2021, 10, 897 .
AMA StyleMelissa Stunnenberg, John van Hamme, Atze Das, Ben Berkhout, Teunis Geijtenbeek. Variations in the Abortive HIV-1 RNA Hairpin Do Not Impede Viral Sensing and Innate Immune Responses. Pathogens. 2021; 10 (7):897.
Chicago/Turabian StyleMelissa Stunnenberg; John van Hamme; Atze Das; Ben Berkhout; Teunis Geijtenbeek. 2021. "Variations in the Abortive HIV-1 RNA Hairpin Do Not Impede Viral Sensing and Innate Immune Responses." Pathogens 10, no. 7: 897.
RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).
Zongliang Gao; Yme Ubeles van der Velden; Minghui Fan; Cynthia Alyssa van der Linden; Monique Vink; Elena Herrera-Carrillo; Ben Berkhout. Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity. Journal of Biological Chemistry 2021, 296, 100026 .
AMA StyleZongliang Gao, Yme Ubeles van der Velden, Minghui Fan, Cynthia Alyssa van der Linden, Monique Vink, Elena Herrera-Carrillo, Ben Berkhout. Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity. Journal of Biological Chemistry. 2021; 296 ():100026.
Chicago/Turabian StyleZongliang Gao; Yme Ubeles van der Velden; Minghui Fan; Cynthia Alyssa van der Linden; Monique Vink; Elena Herrera-Carrillo; Ben Berkhout. 2021. "Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity." Journal of Biological Chemistry 296, no. : 100026.
Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18 base pairs (bp) stem and a 3–5 nucleotides (nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3′ terminus, which generates a uniform 3′ end instead of a 3′ U-tail of variable length. We evaluated the impact of this 3′-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.
Ben Berkhout; Elena Herrera-Carrillo. Design and Evaluation of AgoshRNAs with 3′-Terminal HDV Ribozymes to Enhance the Silencing Activity. Methods in Molecular Biology 2020, 225 -252.
AMA StyleBen Berkhout, Elena Herrera-Carrillo. Design and Evaluation of AgoshRNAs with 3′-Terminal HDV Ribozymes to Enhance the Silencing Activity. Methods in Molecular Biology. 2020; ():225-252.
Chicago/Turabian StyleBen Berkhout; Elena Herrera-Carrillo. 2020. "Design and Evaluation of AgoshRNAs with 3′-Terminal HDV Ribozymes to Enhance the Silencing Activity." Methods in Molecular Biology , no. : 225-252.
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5′ and 3′-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5′ terminus, but the Pol III enzyme generates small RNAs with 3’ U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3′-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.
Ben Berkhout; Zongliang Gao; Elena Herrera-Carrillo. Design and Evaluation of Guide RNA Transcripts with a 3′-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation. Methods in Molecular Biology 2020, 2167, 205 -224.
AMA StyleBen Berkhout, Zongliang Gao, Elena Herrera-Carrillo. Design and Evaluation of Guide RNA Transcripts with a 3′-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation. Methods in Molecular Biology. 2020; 2167 ():205-224.
Chicago/Turabian StyleBen Berkhout; Zongliang Gao; Elena Herrera-Carrillo. 2020. "Design and Evaluation of Guide RNA Transcripts with a 3′-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation." Methods in Molecular Biology 2167, no. : 205-224.
In adherent individuals, antiretroviral therapy (ART) suppresses HIV replication, restores immune function, and prevents the development of AIDS. However, ART is not curative and has to be followed lifelong. Persistence of viral reservoirs forms the major obstacle to an HIV cure. HIV latent reservoirs persist primarily by cell longevity and proliferation, but replenishment by residual virus replication despite ART has been proposed as another potential mechanism of HIV persistence. It is a matter of debate whether different ART regimens are equally potent in suppressing HIV replication. Here, we summarized the current knowledge on the role of ART regimens in HIV persistence, focusing on differences in residual plasma viremia and other virological markers of the HIV reservoir between infected individuals treated with combination ART composed of different antiretroviral drug classes.
Gilles Darcis; Ben Berkhout; Alexander O. Pasternak. Differences in HIV Markers between Infected Individuals Treated with Different ART Regimens: Implications for the Persistence of Viral Reservoirs. Viruses 2020, 12, 489 .
AMA StyleGilles Darcis, Ben Berkhout, Alexander O. Pasternak. Differences in HIV Markers between Infected Individuals Treated with Different ART Regimens: Implications for the Persistence of Viral Reservoirs. Viruses. 2020; 12 (5):489.
Chicago/Turabian StyleGilles Darcis; Ben Berkhout; Alexander O. Pasternak. 2020. "Differences in HIV Markers between Infected Individuals Treated with Different ART Regimens: Implications for the Persistence of Viral Reservoirs." Viruses 12, no. 5: 489.
The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.
Zongliang Gao; Minghui Fan; Atze T Das; Elena Herrera-Carrillo; Ben Berkhout. Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA. Nucleic Acids Research 2020, 48, 5527 -5539.
AMA StyleZongliang Gao, Minghui Fan, Atze T Das, Elena Herrera-Carrillo, Ben Berkhout. Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA. Nucleic Acids Research. 2020; 48 (10):5527-5539.
Chicago/Turabian StyleZongliang Gao; Minghui Fan; Atze T Das; Elena Herrera-Carrillo; Ben Berkhout. 2020. "Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA." Nucleic Acids Research 48, no. 10: 5527-5539.
Plasma viral load (VL) and CD4+ T cell count are widely used as biomarkers of HIV type 1 (HIV-1) replication, pathogenesis, and response to antiretroviral therapy (ART). However, the clinical potential of cell-associated (CA) HIV-1 molecular markers is much less understood. Here, we measured CA HIV-1 RNA and DNA in HIV-infected individuals treated with temporary ART initiated during primary HIV-1 infection. We demonstrate substantial predictive value of CA RNA for (a) the virological and immunological response to early ART, (b) the magnitude and time to viral rebound after discontinuation of early ART, and (c) disease progression in the absence of treatment. Remarkably, when adjusted for CA RNA, plasma VL no longer appeared as an independent predictor of any clinical endpoint in this cohort. The potential of CA RNA as an HIV-1 clinical marker, in particular as a predictive biomarker of virological control after stopping ART, should be explored in the context of HIV-1 curative interventions.
Alexander O. Pasternak; Marlous L. Grijsen; Ferdinand W. Wit; Margreet Bakker; Suzanne Jurriaans; Jan M. Prins; Ben Berkhout. Cell-associated HIV-1 RNA predicts viral rebound and disease progression after discontinuation of temporary early ART. JCI Insight 2020, 5, 1 .
AMA StyleAlexander O. Pasternak, Marlous L. Grijsen, Ferdinand W. Wit, Margreet Bakker, Suzanne Jurriaans, Jan M. Prins, Ben Berkhout. Cell-associated HIV-1 RNA predicts viral rebound and disease progression after discontinuation of temporary early ART. JCI Insight. 2020; 5 (6):1.
Chicago/Turabian StyleAlexander O. Pasternak; Marlous L. Grijsen; Ferdinand W. Wit; Margreet Bakker; Suzanne Jurriaans; Jan M. Prins; Ben Berkhout. 2020. "Cell-associated HIV-1 RNA predicts viral rebound and disease progression after discontinuation of temporary early ART." JCI Insight 5, no. 6: 1.
Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target sites correlated with permanent virus inactivation. To better understand the mechanism underlying HIV inactivation, we analyzed the proviral DNA upon Cas9 attack with gRNA pairs. We observed that dual-gRNA targeting resulted more frequently in target site mutation than fragment excision, while fragment inversion was rarely observed. The frequencies varied for different gRNA combinations without an obvious relationship with the distance between the target sites, indicating that other gRNA and target DNA characteristics influence the DNA cleavage and repair processes.
Caroline S. Binda; Bep Klaver; Ben Berkhout; Atze T. Das. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA. Viruses 2020, 12, 330 .
AMA StyleCaroline S. Binda, Bep Klaver, Ben Berkhout, Atze T. Das. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA. Viruses. 2020; 12 (3):330.
Chicago/Turabian StyleCaroline S. Binda; Bep Klaver; Ben Berkhout; Atze T. Das. 2020. "CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA." Viruses 12, no. 3: 330.
The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.
Franziska Hentzschel; Vera Mitesser; Sabine Anne-Kristin Fraschka; Daria Krzikalla; Elena Herrera Carrillo; Ben Berkhout; Richárd Bártfai; Ann-Kristin Mueller; Dirk Grimm. Gene knockdown in malaria parasites via non-canonical RNAi. Nucleic Acids Research 2019, 48, e2 -e2.
AMA StyleFranziska Hentzschel, Vera Mitesser, Sabine Anne-Kristin Fraschka, Daria Krzikalla, Elena Herrera Carrillo, Ben Berkhout, Richárd Bártfai, Ann-Kristin Mueller, Dirk Grimm. Gene knockdown in malaria parasites via non-canonical RNAi. Nucleic Acids Research. 2019; 48 (1):e2-e2.
Chicago/Turabian StyleFranziska Hentzschel; Vera Mitesser; Sabine Anne-Kristin Fraschka; Daria Krzikalla; Elena Herrera Carrillo; Ben Berkhout; Richárd Bártfai; Ann-Kristin Mueller; Dirk Grimm. 2019. "Gene knockdown in malaria parasites via non-canonical RNAi." Nucleic Acids Research 48, no. 1: e2-e2.
Chen Liang; Ben Berkhout. Editorial overview: Engineering cellular resistance towards the HIV cure. Current Opinion in Virology 2019, 38, 1 .
AMA StyleChen Liang, Ben Berkhout. Editorial overview: Engineering cellular resistance towards the HIV cure. Current Opinion in Virology. 2019; 38 ():1.
Chicago/Turabian StyleChen Liang; Ben Berkhout. 2019. "Editorial overview: Engineering cellular resistance towards the HIV cure." Current Opinion in Virology 38, no. : 1.
Antiretroviral therapy (ART) can effectively suppress ongoing HIV replication and block disease progression, but the infection is never cured due to the persistence of a small pool of latently infected cells hosting integrated replication-competent HIV proviruses. However, the vast majority of HIV proviruses in ART-treated patients are replication-incompetent due to a variety of genetic defects. Most defective proviruses (around 90%) contain large internal deletions or are G-to-A hypermutated, resulting in destruction of most if not all viral open reading frames, which is consistent with the idea that cytotoxic T cells (CTLs) effectively remove cells that produce viral antigens. An intriguing subclass of defective proviruses (around 10%) that are consistently detected in such patients carry a small deletion or a point mutation in a relatively precise and well conserved region near the 5ʹ end of the HIV genome, in the area that encodes the major splice donor (MSD) site and the packaging signal Ѱ in the viral RNA genome. Why this subclass of proviruses is defective has never been properly understood. We now propose a mechanistic scenario for how these MSD-Ѱ mutations can prevent viral protein expression. Based on ample results in literature, we argue that MSD inactivation triggers the activity of the 5ʹ-polyadenylation site, resulting in the production of ultra-short non-protein-coding HIV transcripts.
Atze T. Das; Alexander O. Pasternak; Ben Berkhout. On the generation of the MSD-Ѱ class of defective HIV proviruses. Retrovirology 2019, 16, 1 -5.
AMA StyleAtze T. Das, Alexander O. Pasternak, Ben Berkhout. On the generation of the MSD-Ѱ class of defective HIV proviruses. Retrovirology. 2019; 16 (1):1-5.
Chicago/Turabian StyleAtze T. Das; Alexander O. Pasternak; Ben Berkhout. 2019. "On the generation of the MSD-Ѱ class of defective HIV proviruses." Retrovirology 16, no. 1: 1-5.
RNA interference (RNAi) can be triggered by synthetic small interfering RNAs (siRNAs) or transgene-expressed short hairpin RNAs (shRNAs). Recent evidence indicates that shRNA molecules, with a relatively short stem and small loop, are processed by Argonaute 2 protein (Ago2). We named these molecules AgoshRNA as Ago2 is involved in both the processing and the subsequent mRNA-silencing reaction. This alternative processing route yields only a single guide strand, which thus avoids potential off-target effects induced by the passenger strand of a regular shRNA. We recently described that the introduction of a 5ʹ-terminal purine (A or G) and a mismatch at the bottom of the hairpin enhances the AgoshRNA activity. The critical 5ʹ-terminal nucleotide (nt) represents the +1 position of the transcriptional promoter, which influences the transcriptional efficiency and initiation accuracy as demonstrated for the H1 RNA polymerase (Pol) III promoter. These findings highlight the necessity of considering Pol III requirements in the design of optimized AgoshRNA cassettes. In this study, we report the design and expression of potent AgoshRNAs by two other popular Pol III promoters: U6 and 7SK, which were recently reported to have a distinct transcription profile compared to the H1 promoter. We propose general rules for the design and expression of potent AgoshRNA molecules using Pol III cassettes, which should augment the application of novel AgoshRNA reagents for basic research and therapeutic purposes.
Zongliang Gao; Ben Berkhout; Elena Herrera-Carrillo. Boosting AgoshRNA activity by optimized 5’-terminal nucleotide selection. RNA Biology 2019, 16, 890 -898.
AMA StyleZongliang Gao, Ben Berkhout, Elena Herrera-Carrillo. Boosting AgoshRNA activity by optimized 5’-terminal nucleotide selection. RNA Biology. 2019; 16 (7):890-898.
Chicago/Turabian StyleZongliang Gao; Ben Berkhout; Elena Herrera-Carrillo. 2019. "Boosting AgoshRNA activity by optimized 5’-terminal nucleotide selection." RNA Biology 16, no. 7: 890-898.
Short hairpin RNAs (shRNAs) can induce gene silencing via the RNA interference (RNAi) mechanism. We designed an alternative shRNA molecule with a relatively short base-paired stem that bypasses Dicer and instead is processed by the Argonaute 2 (Ago2) protein into a single guide RNA strand that effectively induces RNAi. We called these molecules AgoshRNAs. Active anti-HIV AgoshRNAs were developed, but their RNAi activity was generally reduced compared with the matching shRNAs. In an attempt to further optimize the AgoshRNA design, we inserted several self-cleaving ribozymes at the 3′ terminus of the transcribed AgoshRNA and evaluated the impact on AgoshRNA processing and activity. The hepatitis delta virus (HDV) ribozyme is efficiently removed from the transcribed AgoshRNAs and generates a uniform 3′ overhang, which translates into the enhanced antiviral activity of these molecules.
Elena Herrera-Carrillo; Zongliang Gao; Ben Berkhout. Influence of a 3′ Terminal Ribozyme on AgoshRNA Biogenesis and Activity. Molecular Therapy - Nucleic Acids 2019, 16, 452 -462.
AMA StyleElena Herrera-Carrillo, Zongliang Gao, Ben Berkhout. Influence of a 3′ Terminal Ribozyme on AgoshRNA Biogenesis and Activity. Molecular Therapy - Nucleic Acids. 2019; 16 ():452-462.
Chicago/Turabian StyleElena Herrera-Carrillo; Zongliang Gao; Ben Berkhout. 2019. "Influence of a 3′ Terminal Ribozyme on AgoshRNA Biogenesis and Activity." Molecular Therapy - Nucleic Acids 16, no. : 452-462.
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.
Gilles Darcis; Caroline S. Binda; Bep Klaver; Elena Herrera-Carrillo; Ben Berkhout; Atze T. Das. The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape. Viruses 2019, 11, 255 .
AMA StyleGilles Darcis, Caroline S. Binda, Bep Klaver, Elena Herrera-Carrillo, Ben Berkhout, Atze T. Das. The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape. Viruses. 2019; 11 (3):255.
Chicago/Turabian StyleGilles Darcis; Caroline S. Binda; Bep Klaver; Elena Herrera-Carrillo; Ben Berkhout; Atze T. Das. 2019. "The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape." Viruses 11, no. 3: 255.
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of self-cleaving ribozymes that facilitate processing of the crRNA transcript to produce the precise guide molecule. Insertion of the 3ʹ-terminal HDV ribozyme boosted the gene editing activity of the CRISPR-Cpf1 system ranging from 1.1 to 5.2 fold. We also demonstrate that this design can enhance CRISPR-based gene activation. We thus generated an improved CRISPR-Cpf1 system for more efficient gene editing and gene regulation.
Zongliang Gao; Elena Herrera-Carrillo; Ben Berkhout. Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA. RNA Biology 2018, 15, 1458 -1467.
AMA StyleZongliang Gao, Elena Herrera-Carrillo, Ben Berkhout. Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA. RNA Biology. 2018; 15 (12):1458-1467.
Chicago/Turabian StyleZongliang Gao; Elena Herrera-Carrillo; Ben Berkhout. 2018. "Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA." RNA Biology 15, no. 12: 1458-1467.
The RNA-guided endonuclease Cas9 (CRISPR-Cas9) genome editing system has been widely used for biomedical research and holds great potential for therapeutic applications in eukaryotes. The conventional vector-based CRISPR-Cas9 delivery system requires two different RNA polymerase promoters for expression of the guide RNA (gRNA) and Cas9 endonuclease. The large size and relative complexity of such CRISPR transgene cassettes impede their broad implementation, especially in gene therapy applications with viral vectors that have a limited packaging capacity. Here, we report the design of a single-promoter-driven CRISPR-Cas9 system that uses the dual-polymerase (Pol II and Pol III) activity of the H1 promoter. This size reduction strategy of the vector insert provides a significant titer advantage in the lentiviral vector over the regular CRISPR system.
Zongliang Gao; Elena Herrera-Carrillo; Ben Berkhout. A Single H1 Promoter Can Drive Both Guide RNA and Endonuclease Expression in the CRISPR-Cas9 System. Molecular Therapy - Nucleic Acids 2018, 14, 32 -40.
AMA StyleZongliang Gao, Elena Herrera-Carrillo, Ben Berkhout. A Single H1 Promoter Can Drive Both Guide RNA and Endonuclease Expression in the CRISPR-Cas9 System. Molecular Therapy - Nucleic Acids. 2018; 14 ():32-40.
Chicago/Turabian StyleZongliang Gao; Elena Herrera-Carrillo; Ben Berkhout. 2018. "A Single H1 Promoter Can Drive Both Guide RNA and Endonuclease Expression in the CRISPR-Cas9 System." Molecular Therapy - Nucleic Acids 14, no. : 32-40.
Nucleotide skew analysis is a versatile method to study the nucleotide composition of RNA/DNA molecules, in particular to reveal characteristic sequence signatures. For instance, skew analysis of the nucleotide bias of several viral RNA genomes indicated that it is enriched in the unpaired, single-stranded genome regions, thus creating an even more striking virus-specific signature. The comparison of skew graphs for many virus isolates or families is difficult, time-consuming, and nonquantitative. Here, we present a procedure for a more simple identification of similarities and dissimilarities between nucleotide skew data of coronavirus, flavivirus, picornavirus, and HIV-1 RNA genomes. Window and step sizes were normalized to correct for differences in length of the viral genome. Cumulative skew data are converted into pairwise Euclidean distance matrices, which can be presented as neighbor-joining trees. We present skew value trees for the four virus families and show that closely related viruses are placed in small clusters. Importantly, the skew value trees are similar to the trees constructed by a “classical” model of evolutionary nucleotide substitution. Thus, we conclude that the simple calculation of Euclidean distances between nucleotide skew data allows an easy and quantitative comparison of characteristic sequence signatures of virus genomes. These results indicate that the Euclidean distance analysis of nucleotide skew data forms a nice addition to the virology toolbox.
Formijn Van Hemert; Maarten Jebbink; Andries Van Der Ark; Frits Scholer; Ben Berkhout. Euclidean Distance Analysis Enables Nucleotide Skew Analysis in Viral Genomes. Computational and Mathematical Methods in Medicine 2018, 2018, 1 -9.
AMA StyleFormijn Van Hemert, Maarten Jebbink, Andries Van Der Ark, Frits Scholer, Ben Berkhout. Euclidean Distance Analysis Enables Nucleotide Skew Analysis in Viral Genomes. Computational and Mathematical Methods in Medicine. 2018; 2018 ():1-9.
Chicago/Turabian StyleFormijn Van Hemert; Maarten Jebbink; Andries Van Der Ark; Frits Scholer; Ben Berkhout. 2018. "Euclidean Distance Analysis Enables Nucleotide Skew Analysis in Viral Genomes." Computational and Mathematical Methods in Medicine 2018, no. : 1-9.
Volker Thiel; Ben Berkhout. Virus Research starts publishing the new “Virus Research Consortia Series”. Virus Research 2018, 257, 119 .
AMA StyleVolker Thiel, Ben Berkhout. Virus Research starts publishing the new “Virus Research Consortia Series”. Virus Research. 2018; 257 ():119.
Chicago/Turabian StyleVolker Thiel; Ben Berkhout. 2018. "Virus Research starts publishing the new “Virus Research Consortia Series”." Virus Research 257, no. : 119.
Broadly neutralizing antibodies (bNAbs) such as PGDM1400 show promise as prophylactic and therapeutic agents against HIV-1. Human immune system (HIS) mice were passively immunized with different doses of PGDM1400 and challenged 24 hours later with a high dose of HIV-1JRCSF. We found that PGDM1400 provided protection against HIV-1 challenge in a concentration dependent manner and that the protective concentration in blood was approximately 75-fold higher than the in vitro 50% inhibitory concentration (IC50). The results demonstrate that PGDM1400 might be a promising component of strategies to prevent HIV-1 infection, and provide support for the pursuit of vaccines that induce PGDM1400-like bNAbs.
Yme U. Van Der Velden; Julien Villaudy; Esther Siteur - Van Rijnstra; Cynthia A. Van Der Linden; Esmay Frankin; Kees Weijer; Edith Schermer; Monique A. Vink; Ben Berkhout; Rogier W. Sanders; Marit J. Van Gils; Esmay Franklin. Short Communication: Protective Efficacy of Broadly Neutralizing Antibody PGDM1400 Against HIV-1 Challenge in Humanized Mice. AIDS Research and Human Retroviruses 2018, 34, 790 -793.
AMA StyleYme U. Van Der Velden, Julien Villaudy, Esther Siteur - Van Rijnstra, Cynthia A. Van Der Linden, Esmay Frankin, Kees Weijer, Edith Schermer, Monique A. Vink, Ben Berkhout, Rogier W. Sanders, Marit J. Van Gils, Esmay Franklin. Short Communication: Protective Efficacy of Broadly Neutralizing Antibody PGDM1400 Against HIV-1 Challenge in Humanized Mice. AIDS Research and Human Retroviruses. 2018; 34 (9):790-793.
Chicago/Turabian StyleYme U. Van Der Velden; Julien Villaudy; Esther Siteur - Van Rijnstra; Cynthia A. Van Der Linden; Esmay Frankin; Kees Weijer; Edith Schermer; Monique A. Vink; Ben Berkhout; Rogier W. Sanders; Marit J. Van Gils; Esmay Franklin. 2018. "Short Communication: Protective Efficacy of Broadly Neutralizing Antibody PGDM1400 Against HIV-1 Challenge in Humanized Mice." AIDS Research and Human Retroviruses 34, no. 9: 790-793.
RNA interference (RNAi) was discovered in plants where it functions as the main antiviral pathway and this antiviral role was subsequently extended to invertebrates. But it remained hotly debated whether RNAi fulfils a similar role in mammals that already have a potent innate immune system based on interferon and an elaborate adaptive immune system. On the one hand, mammalian cells do encode most of the RNAi machinery, but this could be used exclusively to control cellular gene expression via micro RNAs (miRNAs). But on the other hand, virus-derived small interfering RNAs, the hallmark of RNAi involvement, could not be readily detected upon virus infection of mammalian cells. However, recent studies have indicated that these signature molecules are generated in virus-infected embryonic cell types of mammals and that viruses actively suppress such responses by means of potent RNAi suppressor proteins. Thus, the tide seems to be changing in favor of RNAi as accessory antiviral defense mechanism in humans. Intriguingly, recent studies indicate that insects have also developed an additional innate immune system that collaborates with the RNAi response in the fight against invading viral pathogens. Thus, the presence of multiple antiviral response mechanisms seems standard outside the plant world and we will specifically discuss the interactions between these antiviral programs.
Ben Berkhout. RNAi-mediated antiviral immunity in mammals. Current Opinion in Virology 2018, 32, 9 -14.
AMA StyleBen Berkhout. RNAi-mediated antiviral immunity in mammals. Current Opinion in Virology. 2018; 32 ():9-14.
Chicago/Turabian StyleBen Berkhout. 2018. "RNAi-mediated antiviral immunity in mammals." Current Opinion in Virology 32, no. : 9-14.