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Asset Tag: Circular consensus sequencing

October 23, 2019

Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity.

Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal repeats (mTRs), sequences that were previously thought to be required for functional self-complementary AAV vectors. The use of shDNAs as artificial mTRs opens the door to engineering a new generation of AAV vectors with improved potency, genetic stability, and safety for both preclinical studies and human gene therapy. Published by Elsevier Inc.

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October 23, 2019

CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions.

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion (“ScanDel”). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation. Copyright © 2017 American Society of Human Genetics. All rights reserved.

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October 23, 2019

Structural determination of the broadly reactive anti-IGHV1-69 anti-idiotypic antibody G6 and its idiotope.

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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October 23, 2019

Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.

Recent advances using CRISPR-Cas9 approaches have dramatically enhanced the ease for genetic manipulation in rodents. Notwithstanding, the methods to deliver nucleic acids into pre-implantation embryos have hardly changed since the original description of mouse transgenesis more than 30 years ago. Here we report a novel strategy to generate genetically modified mice by transduction of CRISPR-Cas9 components into pre-implantation mouse embryos via recombinant adeno-associated viruses (rAAVs). Using this approach, we efficiently generated a variety of targeted mutations in explanted embryos, including indel events produced by non-homologous end joining and tailored mutations using homology-directed repair. We also achieved gene modification in vivo by direct delivery of rAAV particles into the oviduct of pregnant females. Our approach greatly simplifies the generation of genetically modified mice and, more importantly, opens the door for streamlined gene editing in other mammalian species.

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October 23, 2019

Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing.

Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease dependency. Furthermore, de novo sequences can be inserted without the requirement of homologous recombination. Such reprogramming would enable efficient editing in quiescent cells (e.g., neurons) with an attractive safety profile for translational therapies. In this study, we applied a novel molecular-barcoded screening assay to investigate RNA trans-splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determining trans-splicing are reproducible regardless of the screening system. With this screening, we have located the most permissive trans-splicing sequences targeting an intron in the Synapsin I gene. Using viral vectors, we were able to splice full-length fluorophores into the mRNA while retaining very low off-target expression. Furthermore, this approach also showed evidence of functionality in the mouse striatum. However, in its current form, the trans-splicing events are stochastic and the overall activity lower than would be required for therapies targeting loss-of-function mutations. Nevertheless, the herein described barcode-based screening assay provides a unique possibility to screen and map large libraries in single animals or cell assays with very high precision.© 2018 Davidsson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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October 23, 2019

Bioengineered viral platform for intramuscular passive vaccine delivery to human skeletal muscle.

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.

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October 23, 2019

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3′-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

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October 23, 2019

Dynamics of coral-associated microbiomes during a thermal bleaching event.

Coral-associated microorganisms play an important role in their host fitness and survival. A number of studies have demonstrated connections between thermal tolerance in corals and the type/relative abundance of Symbiodinium they harbor. More recently, the shifts in coral-associated bacterial profiles were also shown to be linked to the patterns of coral heat tolerance. Here, we investigated the dynamics of Porites lutea-associated bacterial and algal communities throughout a natural bleaching event, using full-length 16S rRNA and internal transcribed spacer sequences (ITS) obtained from PacBio circular consensus sequencing. We provided evidence of significant changes in the structure and diversity of coral-associated microbiomes during thermal stress. The balance of the symbiosis shifted from a predominant association between corals and Gammaproteobacteria to a predominance of Alphaproteobacteria and to a lesser extent Betaproteobacteria following the bleaching event. On the contrary, the composition and diversity of Symbiodinium communities remained unaltered throughout the bleaching event. It appears that the switching and/or shuffling of Symbiodinium types may not be the primary mechanism used by P. lutea to cope with increasing seawater temperature. The shifts in the structure and diversity of associated bacterial communities may contribute more to the survival of the coral holobiont under heat stress.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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October 23, 2019

An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage.

Although engineered nucleases can efficiently cleave intracellular DNA at desired target sites, major concerns remain on potential ‘off-target’ cleavage that may occur throughout the genome. We developed an online tool: predicted report of genome-wide nuclease off-target sites (PROGNOS) that effectively identifies off-target sites. The initial bioinformatics algorithms in PROGNOS were validated by predicting 44 of 65 previously confirmed off-target sites, and by uncovering a new off-target site for the extensively studied zinc finger nucleases (ZFNs) targeting C-C chemokine receptor type 5. Using PROGNOS, we rapidly interrogated 128 potential off-target sites for newly designed transcription activator-like effector nucleases containing either Asn-Asn (NN) or Asn-Lys (NK) repeat variable di-residues (RVDs) and 3- and 4-finger ZFNs, and validated 13 bona fide off-target sites for these nucleases by DNA sequencing. The PROGNOS algorithms were further refined by incorporating additional features of nuclease-DNA interactions and the newly confirmed off-target sites into the training set, which increased the percentage of bona fide off-target sites found within the top PROGNOS rankings. By identifying potential off-target sites in silico, PROGNOS allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of engineered nucleases for genome editing applications.

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October 23, 2019

High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system.

Coral reefs are a complex ecosystem consisting of coral animals and a vast array of associated symbionts including the dinoflagellate Symbiodinium, fungi, viruses and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their fundamental roles in fitness and survival of the host animal. The scleractinian coral Porites lutea is one of the dominant reef-builders in the Indo-West Pacific. Currently, very little is known about the composition and structure of bacterial communities across P. lutea reefs. The purpose of this study is twofold: to demonstrate the advantages of using PacBio circular consensus sequencing technology in microbial community studies and to investigate the diversity and structure of P. lutea-associated microbiome in the Indo-Pacific. This is the first metagenomic study of marine environmental samples that utilises the PacBio sequencing system to capture full-length 16S rRNA sequences. We observed geographically distinct coral-associated microbial profiles between samples from the Gulf of Thailand and Andaman Sea. Despite the geographical and environmental impacts on the coral-host interactions, we identified a conserved community of bacteria that were present consistently across diverse reef habitats. Finally, we demonstrated the superior performance of full-length 16S rRNA sequences in resolving taxonomic uncertainty of coral associates at the species level.

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October 23, 2019

Bioengineered AAV capsids with combined high human liver transduction in vivo and unique humoral seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications. Copyright © 2017. Published by Elsevier Inc.

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October 23, 2019

Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors.

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September 22, 2019

HIV-1 infection of primary CD4(+) T cells regulates the expression of specific HERV-K (HML-2) elements.

Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathologic states, such as viral infections and certain cancers, coincide with ERV expression suggesting transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic.Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1 infected primary human CD4(+) T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read Single Molecule Real-Time sequencing. We show that three HML-2 proviruses, 6q25.1, 8q24.3, and 19q13.42 are up-regulated on average between 3- and 5-fold in HIV-1 infected CD4(+) T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication.In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4(+) T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication.Importance Endogenous retroviruses inhabit big portions of our genome. And although they are mainly inert some of the evolutionarily younger members maintain the ability to express both RNA as well as proteins. We have developed an approach using long-read SMRT sequencing that produces long reads, that provides us with ability to obtain detailed and accurate HERV-K HML-2 expression profiles. We have now applied this approach to study HERV-K expression in the presence and absence of productive HIV-1 infection of primary human CD4(+) T cells. In addition to using SMRT sequencing, our strategy also includes the magnetic selection of the infected cells so that levels of background expression due to uninfected cells are kept at a minimum. The results in this manuscript provide the blueprint for in-depth studies of the interactions of the authentic upregulated HERV-K HML-2 elements and HIV-1. Copyright © 2017 American Society for Microbiology.

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September 22, 2019

Global transcript structure resolution of high gene density genomes through multi-platform data integration.

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

Subaerial biofilms on granitic historic buildings: microbial diversity and development of phototrophic multi-species cultures.

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.

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