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April 21, 2020

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

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April 21, 2020

The Single-molecule long-read sequencing of Scylla paramamosain.

Scylla paramamosain is an important aquaculture crab, which has great economical and nutritional value. To the best of our knowledge, few full-length crab transcriptomes are available. In this study, a library composed of 12 different tissues including gill, hepatopancreas, muscle, cerebral ganglion, eyestalk, thoracic ganglia, intestine, heart, testis, ovary, sperm reservoir, and hemocyte was constructed and sequenced using Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology. A total of 284803 full-length non-chimeric reads were obtained, from which 79005 high-quality unique transcripts were obtained after error correction and sequence clustering and redundant. Additionally, a total of 52544 transcripts were annotated against protein database (NCBI nonredundant, Swiss-Prot, KOG, and KEGG database). A total of 23644 long non-coding RNAs (lncRNAs) and 131561 simple sequence repeats (SSRs) were identified. Meanwhile, the isoforms of many genes were also identified in this study. Our study provides a rich set of full-length cDNA sequences for S. paramamosain, which will greatly facilitate S. paramamosain research.

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April 21, 2020

Genetic basis of functional variability in adhesion G protein-coupled receptors.

The enormous sizes of adhesion G protein-coupled receptors (aGPCRs) go along with complex genomic exon-intron architectures giving rise to multiple mRNA variants. There is a need for a comprehensive catalog of aGPCR variants for proper evaluation of the complex functions of aGPCRs found in structural, in vitro and animal model studies. We used an established bioinformatics pipeline to extract, quantify and visualize mRNA variants of aGPCRs from deeply sequenced transcriptomes. Data analysis showed that aGPCRs have multiple transcription start sites even within introns and that tissue-specific splicing is frequent. On average, 19 significantly expressed transcript variants are derived from a given aGPCR gene. The domain architecture of the N terminus encoded by transcript variants often differs and N termini without or with an incomplete seven-helix transmembrane anchor as well as separate seven-helix transmembrane domains are frequently derived from aGPCR genes. Experimental analyses of selected aGPCR transcript variants revealed marked functional differences. Our analysis has an impact on a rational design of aGPCR constructs for structural analyses and gene-deficient mouse lines and provides new support for independent functions of both, the large N terminus and the transmembrane domain of aGPCRs.

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April 21, 2020

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

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April 21, 2020

Complete assembly of the Leishmania donovani (HU3 strain) genome and transcriptome annotation.

Leishmania donovani is a unicellular parasite that causes visceral leishmaniasis, a fatal disease in humans. In this study, a complete assembly of the genome of L. donovani is provided. Apart from being the first published genome of this strain (HU3), this constitutes the best assembly for an L. donovani genome attained to date. The use of a combination of sequencing platforms enabled to assemble, without any sequence gap, the 36 chromosomes for this species. Additionally, based on this assembly and using RNA-seq reads derived from poly-A?+?RNA, the transcriptome for this species, not yet available, was delineated. Alternative SL addition sites and heterogeneity in the poly-A addition sites were commonly observed for most of the genes. After a complete annotation of the transcriptome, 2,410 novel transcripts were defined. Additionally, the relative expression for all transcripts present in the promastigote stage was determined. Events of cis-splicing have been documented to occur during the maturation of the transcripts derived from genes LDHU3_07.0430 and LDHU3_29.3990. The complete genome assembly and the availability of the gene models (including annotation of untranslated regions) are important pieces to understand how differential gene expression occurs in this pathogen, and to decipher phenotypic peculiarities like tissue tropism, clinical disease, and drug susceptibility.

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April 21, 2020

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

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April 21, 2020

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

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April 21, 2020

Metatranscriptomic evidence for classical and RuBisCO-mediated CO2 reduction to methane facilitated by direct interspecies electron transfer in a methanogenic system.

In a staged anaerobic fluidized-bed ceramic membrane bioreactor, metagenomic and metatranscriptomic analyses were performed to decipher the microbial interactions on the granular activated carbon. Metagenome bins, representing the predominating microbes in the bioreactor: syntrophic propionate-oxidizing bacteria (SPOB), acetoclastic Methanothrix concilii, and exoelectrogenic Geobacter lovleyi, were successfully recovered for the reconstruction and analysis of metabolic pathways involved in the transformation of fatty acids to methane. In particular, SPOB degraded propionate into acetate, which was further converted into methane and CO2 by M. concilii via the acetoclastic methanogenesis. Concurrently, G. lovleyi oxidized acetate into CO2, releasing electrons into the extracellular environment. By accepting these electrons through direct interspecies electron transfer (DIET), M. concilii was capable of performing CO2 reduction for further methane formation. Most notably, an alternative RuBisCO-mediated CO2 reduction (the reductive hexulose-phosphate (RHP) pathway) is transcriptionally-active in M. concilii. This RHP pathway enables M. concilii dominance and energy gain by carbon fixation and methanogenesis, respectively via a methyl-H4MPT intermediate, constituting the third methanogenesis route. The complete acetate reduction (2 mole methane formation/1 mole acetate consumption), coupling of acetoclastic methanogenesis and two CO2 reduction pathways, are thermodynamically favorable even under very low substrate condition (down to to 10-5?M level). Such tight interactions via both mediated and direct interspecies electron transfer (MIET and DIET), induced by the conductive GAC promote the overall efficiency of bioenergy processes.

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April 21, 2020

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

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April 21, 2020

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

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April 21, 2020

Extensive intraspecific gene order and gene structural variations in upland cotton cultivars.

Multiple cotton genomes (diploid and tetraploid) have been assembled. However, genomic variations between cultivars of allotetraploid upland cotton (Gossypium hirsutum L.), the most widely planted cotton species in the world, remain unexplored. Here, we use single-molecule long read and Hi-C sequencing technologies to assemble genomes of the two upland cotton cultivars TM-1 and zhongmiansuo24 (ZM24). Comparisons among TM-1 and ZM24 assemblies and the genomes of the diploid ancestors reveal a large amount of genetic variations. Among them, the top three longest structural variations are located on chromosome A08 of the tetraploid upland cotton, which account for ~30% total length of this chromosome. Haplotype analyses of the mapping population derived from these two cultivars and the germplasm panel show suppressed recombination rates in this region. This study provides additional genomic resources for the community, and the identified genetic variations, especially the reduced meiotic recombination on chromosome A08, will help future breeding.

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April 21, 2020

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia.

IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

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April 21, 2020

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes.

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.

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April 21, 2020

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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