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Wednesday, 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…

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Wednesday, 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…

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Sunday, September 22, 2019

Searching for convergent pathways in autism spectrum disorders: insights from human brain transcriptome studies.

Autism spectrum disorder (ASD) is one of the most heritable neuropsychiatric conditions. The complex genetic landscape of the disorder includes both common and rare variants at hundreds of genetic loci. This marked heterogeneity has thus far hampered efforts to develop genetic diagnostic panels and targeted pharmacological therapies. Here, we give an overview of the current literature on the genetic basis of ASD, and review recent human brain transcriptome studies and their role in identifying convergent pathways downstream of the heterogeneous genetic variants. We also discuss emerging evidence on the involvement of non-coding genomic regions and non-coding RNAs in ASD.

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Sunday, September 22, 2019

The transcriptome of human pluripotent stem cells.

Human Embryonic Stem Cells (hESCs) are in vitro derivatives of the inner cell mass of the blastocyst and are characterized by an undifferentiated and pluripotent state that can be perpetuated in time, indefinitely. hESCs provide a unique opportunity to both dissect the molecular mechanisms that are predisposed to the maintenance of pluripotency and model the ability to initiate differentiation and cell commitment within the developing embryo. To fully understand these mechanisms, it is necessary to accurately identify the specific transcriptome of hESCs. Many distinct gene annotation methods, such as cDNA and EST sequencing and RNA-Seq, have been used to identify…

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Sunday, September 22, 2019

Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae.

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of…

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Sunday, 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…

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Sunday, September 22, 2019

The state of long non-coding RNA biology.

Transcriptomic studies have demonstrated that the vast majority of the genomes of mammals and other complex organisms is expressed in highly dynamic and cell-specific patterns to produce large numbers of intergenic, antisense and intronic long non-protein-coding RNAs (lncRNAs). Despite well characterized examples, their scaling with developmental complexity, and many demonstrations of their association with cellular processes, development and diseases, lncRNAs are still to be widely accepted as major players in gene regulation. This may reflect an underappreciation of the extent and precision of the epigenetic control of differentiation and development, where lncRNAs appear to have a central role, likely as…

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Sunday, September 22, 2019

A near complete snapshot of the Zea mays seedling transcriptome revealed from ultra-deep sequencing.

RNA-sequencing (RNA-seq) enables in-depth exploration of transcriptomes, but typical sequencing depth often limits its comprehensiveness. In this study, we generated nearly 3 billion RNA-Seq reads, totaling 341 Gb of sequence, from a Zea mays seedling sample. At this depth, a near complete snapshot of the transcriptome was observed consisting of over 90% of the annotated transcripts, including lowly expressed transcription factors. A novel hybrid strategy combining de novo and reference-based assemblies yielded a transcriptome consisting of 126,708 transcripts with 88% of expressed known genes assembled to full-length. We improved current annotations by adding 4,842 previously unannotated transcript variants and many…

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Sunday, September 22, 2019

RNA sequencing (RNA-Seq) reveals extremely low levels of reticulocyte-derived globin gene transcripts in peripheral blood from horses (Equus caballus) and cattle (Bos taurus).

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of…

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Sunday, September 22, 2019

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis.

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (?123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3’UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1-3 in the ?123…

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Sunday, September 22, 2019

SMRT-Cappable-seq reveals complex operon variants in bacteria.

Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5′ and 3′ ends of operons. Here we develop a methodology called SMRT-Cappable-seq that combines the isolation of un-fragmented primary transcripts with single-molecule long read sequencing. Applied to E. coli, this technology results in an accurate definition of the transcriptome with 34% of known operons from RegulonDB being extended by at least one gene. Furthermore, 40% of transcription termination sites have…

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Sunday, September 22, 2019

PRAPI: post-transcriptional regulation analysis pipeline for Iso-Seq.

The single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) based on Pacific Bioscience (PacBio) platform has received increasing attention for its ability to explore full-length isoforms. Thus, comprehensive tools for Iso-Seq bioinformatics analysis are extremely useful. Here, we present a one-stop solution for Iso-Seq analysis, called PRAPI to analyze alternative transcription initiation (ATI), alternative splicing (AS), alternative cleavage and polyadenylation (APA), natural antisense transcripts (NAT), and circular RNAs (circRNAs) comprehensively. PRAPI is capable of combining Iso-Seq full-length isoforms with short read data, such as RNA-Seq or polyadenylation site sequencing (PAS-seq) for differential expression analysis of NAT, AS, APA and circRNAs. Furthermore, PRAPI…

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Sunday, September 22, 2019

ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm.

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of…

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Sunday, September 22, 2019

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data…

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Sunday, September 22, 2019

Differential TGFß pathway targeting by miR-122 in humans and mice affects liver cancer metastasis.

Downregulation of a predominantly hepatocyte-specific miR-122 is associated with human liver cancer metastasis, whereas miR-122-deficient mice display normal liver function. Here we show a functional conservation of miR-122 in the TGFß pathway: miR-122 target site is present in the mouse but not human TGFßR1, whereas a noncanonical target site is present in the TGFß1 5’UTR in humans and other primates. Experimental switch of the miR-122 target between the receptor TGFßR1 and the ligand TGFß1 changes the metastatic properties of mouse and human liver cancer cells. High expression of TGFß1 in human primary liver tumours is associated with poor survival. We…

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