Genomic Position Research Articles

Canis MitoSNP database: a functional tool useful for comparative analyses of human and canine mitochondrial genomes.

Canis MitoSNP is a tool allowing assignment of each mitochondrial genomic position a corresponding position in the mitochondrial gene and in the structure of tRNA, rRNA, and protein. The main aim of this bioinformatic tool was to use data from other bioinformatic tools (TMHMM, SOPMA, tRNA-SCAN, RNAfold, ConSurf) for dog and human mitochondrial genes in order to shorten the time necessary for the analysis of the whole genome single nucleotide polymorphism (SNP) as well as amino acid and protein analyses. Each position in the canine mitochondrial genome is assigned a position in genes, in codons, an amino acid position in proteins, or a position in tRNA or rRNA molecules. Therefore, a user analysing changes in the canine and human mitochondrial genome does not need to extract the sequences of individual genes from the mitochondrial genome for analysis and there is no need to rewrite them into amino acid sequences to assess whether the change is synonymous or nonsynonymous. Canis mitoSNP allows the comparison between the human and canine mitochondrial genomes as well. The Clustal W alignment of the dog and human mitochondrial DNA reference sequences for each gene obtained from GenBank (NC_002008.4 dog, NC_012920.1 human) was performed in order to determine which position in the canine mitochondrial genome corresponds to the position in the human mitochondrial genome. This function may be useful for the comparative analyses. The tool is available at: https://canismitosnp.pl .

Transposable elements in mammalian chromatin organization.

Transposable elements (TEs) are mobile DNA elements that comprise almost 50% of mammalian genomic sequence. TEs are capable of making additional copies of themselves that integrate into new positions in host genomes. This unique property has had an important impact on mammalian genome evolution and on the regulation of gene expression because TE-derived sequences can function as cis-regulatory elements such as enhancers, promoters and silencers. Now, advances in our ability to identify and characterize TEs have revealed that TE-derived sequences also regulate gene expression by both maintaining and shaping 3D genome architecture. Studies are revealing how TEs contribute raw sequence that can give rise to the structures that shape chromatin organization, and thus gene expression, allowing for species-specific genome innovation and evolutionary novelty.

Global analysis of the yeast knockout phenome.

Genome-wide phenotypic screens in the budding yeast Saccharomyces cerevisiae, enabled by its knockout collection, have produced the largest, richest, and most systematic phenotypic description of any organism. However, integrative analyses of this rich data source have been virtually impossible because of the lack of a central data repository and consistent metadata annotations. Here, we describe the aggregation, harmonization, and analysis of ~14,500 yeast knockout screens, which we call Yeast Phenome. Using this unique dataset, we characterized two unknown genes (YHR045W and YGL117W) and showed that tryptophan starvation is a by-product of many chemical treatments. Furthermore, we uncovered an exponential relationship between phenotypic similarity and intergenic distance, which suggests that gene positions in both yeast and human genomes are optimized for function.

The impact of local genomic properties on the evolutionary fate of genes.

Functionally indispensable genes are likely to be retained and otherwise to be lost during evolution. This evolutionary fate of a gene can also be affected by factors independent of gene dispensability, including the mutability of genomic positions, but such features have not been examined well. To uncover the genomic features associated with gene loss, we investigated the characteristics of genomic regions where genes have been independently lost in multiple lineages. With a comprehensive scan of gene phylogenies of vertebrates with a careful inspection of evolutionary gene losses, we identified 813 human genes whose orthologs were lost in multiple mammalian lineages: designated 'elusive genes.' These elusive genes were located in genomic regions with rapid nucleotide substitution, high GC content, and high gene density. A comparison of the orthologous regions of such elusive genes across vertebrates revealed that these features had been established before the radiation of the extant vertebrates approximately 500 million years ago. The association of human elusive genes with transcriptomic and epigenomic characteristics illuminated that the genomic regions containing such genes were subject to repressive transcriptional regulation. Thus, the heterogeneous genomic features driving gene fates toward loss have been in place and may sometimes have relaxed the functional indispensability of such genes. This study sheds light on the complex interplay between gene function and local genomic properties in shaping gene evolution that has persisted since the vertebrate ancestor.

MosSCI-mediated exogenous gene expression is modulated by genomic positioning.

Although the Mos1-mediated single-copy insertion (MosSCI) technique has been widely used to generate stable transgenic Caenorhabditis elegans strains, the link between stability of expression and integration site still needs to be explored. Here, experimental evidence is provided that transgenes are not able to match the level of transcription of their native counterpart, and that insertions at certain locations can result in an external stress-mediated increase in expression. Insertion site ttTi5605 on chromosome II was shown to be a superior location, at least when introducing reproduction related genes. Thus, this study provides a reference for the selection of an optimal site for MosSCI which provides acceptable expression performance whilst minimizing undesirable secondary effects.

New Insights in 9q21.13 Microdeletion Syndrome: Genotype–Phenotype Correlation of 28 Patients

The implementation of array comparative genomic hybridisation (array-CGH) allows us to describe new microdeletion/microduplication syndromes which were previously not identified. 9q21.13 microdeletion syndrome is a genetic condition due to the loss of a critical genomic region of approximately 750kb and includes several genes, such as RORB and TRPM6. Here, we report a case of a 7-year-old boy affected by 9q21.13 microdeletion syndrome. He presents with global developmental delay, intellectual disability, autistic behaviour, seizures and facial dysmorphism. Moreover, he has severe myopia, which was previously reported in only another patient with 9q21.13 deletion, and brain anomalies which were never described before in 9q21.13 microdeletion syndrome. We also collect 17 patients from a literature search and 10 cases from DECIPHER database with a total number of 28 patients (including our case). In order to better investigate the four candidate genes RORB, TRPM6, PCSK5, and PRUNE2 for neurological phenotype, we make, for the first time, a classification in four groups of all the collected 28 patients. This classification is based both on the genomic position of the deletions included in the 9q21.3 locus deleted in our patient and on the different involvement of the four-candidate gene. In this way, we compare the clinical problems, the radiological findings, and the dysmorphic features of each group and of all the 28 patients in our article. Moreover, we perform the genotype-phenotype correlation of the 28 patients to better define the syndromic spectrum of 9q21.13 microdeletion syndrome. Finally, we propose a baseline ophthalmological and neurological monitoring of this syndrome.

Assessing the role of REM13, REM34 and REM46 during the transition to the reproductive phase in Arabidopsis thaliana

REM (reproductive meristem) transcription factors have been proposed as regulators of plant reproductive development mainly based on their specific expression patterns in reproductive structures, but their roles are still largely unknown probably because of their redundancy. We selected three REM genes (REM13, REM34 and REM46) for functional analysis, based on their genome position and/or co-expression data.Our results suggest that these genes have a role in flowering time regulation and may modulate cell cycle progression. In addition, protein interaction experiments revealed that REM34 and REM46 interact with each other, suggesting that they might work cooperatively to regulate cell division during inflorescence meristem commitment.Previous attempts of using co-expression data as a guide for functional analysis of REMs were limited by the transcriptomic data available at the time. Our results uncover previously unknown functions of three members of the REM family of Arabidopsis thaliana and open the door to more comprehensive studies of the REM family, where the combination of co-expression analysis followed by functional studies might contribute to uncovering the biological roles of these proteins and the relationship among them.

Transcription factor expression is the main determinant of variability in gene co‐activity

Many genes are co‐expressed and form genomic domains of coordinated gene activity. However, the regulatory determinants of domain co‐activity remain unclear. Here, we leverage human individual variation in gene expression to characterize the co‐regulatory processes underlying domain co‐activity and systematically quantify their effect sizes. We employ transcriptional decomposition to extract from RNA expression data an expression component related to co‐activity revealed by genomic positioning. This strategy reveals close to 1,500 co‐activity domains, covering most expressed genes, of which the large majority are invariable across individuals. Focusing specifically on domains with high variability in co‐activity reveals that contained genes have a higher sharing of eQTLs, a higher variability in enhancer interactions, and an enrichment of binding by variably expressed transcription factors, compared to genes within non‐variable domains. Through careful quantification of the relative contributions of regulatory processes underlying co‐activity, we find transcription factor expression levels to be the main determinant of gene co‐activity. Our results indicate that distal trans effects contribute more than local genetic variation to individual variation in co‐activity domains.

Mapping of QTLs for morphophysiological and yield traits under water-deficit stress and well-watered conditions in maize.

Maize productivity is significantly impacted by drought; therefore, improvement of drought tolerance is a critical goal in maize breeding. To achieve this, a better understanding of the genetic basis of drought tolerance is necessary. Our study aimed to identify genomic regions associated with drought tolerance-related traits by phenotyping a mapping population of recombinant inbred lines (RILs) for two seasons under well-watered (WW) and water-deficit (WD) conditions. We also used single nucleotide polymorphism (SNP) genotyping through genotyping-by-sequencing to map these regions and attempted to identify candidate genes responsible for the observed phenotypic variation. Phenotyping of the RILs population revealed significant variability in most of the traits, with normal frequency distributions, indicating their polygenic nature. We generated a linkage map using 1,241 polymorphic SNPs distributed over 10 chromosomes (chrs), covering a total genetic distance of 5,471.55cM. We identified 27 quantitative trait loci (QTLs) associated with various morphophysiological and yield-related traits, with 13 QTLs identified under WW conditions and 12 under WD conditions. We found one common major QTL (qCW2-1) for cob weight and a minor QTL (qCH1-1) for cob height that were consistently identified under both water regimes. We also detected one major and one minor QTL for the Normalized Difference Vegetation Index (NDVI) trait under WD conditions on chr 2, bin 2.10. Furthermore, we identified one major QTL (qCH1-2) and one minor QTL (qCH1-1) on chr 1 that were located at different genomic positions to those identified in earlier studies. We found co-localized QTLs for stomatal conductance and grain yield on chr 6 (qgs6-2 and qGY6-1), while co-localized QTLs for stomatal conductance and transpiration rate were identified on chr 7 (qgs7-1 and qTR7-1). We also attempted to identify the candidate genes responsible for the observed phenotypic variation; our analysis revealed that the major candidate genes associated with QTLs detected under water deficit conditions were related to growth and development, senescence, abscisic acid (ABA) signaling, signal transduction, and transporter activity in stress tolerance. The QTL regions identified in this study may be useful in designing markers that can be utilized in marker-assisted selection breeding. In addition, the putative candidate genes can be isolated and functionally characterized so that their role in imparting drought tolerance can be more fully understood.

Leveraging base-pair mammalian constraint to understand genetic variation and human disease.

Thousands of genomic regions have been associated with heritable human diseases, but attempts to elucidate biological mechanisms are impeded by an inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function, agnostic to cell type or disease mechanism. Single-base phyloP scores from 240 mammals identified 3.3% of the human genome as significantly constrained and likely functional. We compared phyloP scores to genome annotation, association studies, copy-number variation, clinical genetics findings, and cancer data. Constrained positions are enriched for variants that explain common disease heritability more than other functional annotations. Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Efficient Colored de Bruijn Graph for Indexing Reads.

The colored de Bruijn graph is a variation of the de Bruijn graph that has recently been utilized for indexing sequencing reads. Although state-of-the-art methods have achieved small index sizes, they produce many read-incoherent paths that tend to cover the same regions in the source genome sequence. To solve this problem, we propose an accurate coloring method that can reduce the generation of read-incoherent paths by utilizing different colors for a single read depending on the position in the read, which reduces ambiguous coloring in cases where a node has two successors, and both of the successors have the same color. To avoid having to memorize the order of the colors, we utilize a hash function to generate and reproduce the series of colors from the initial color and then apply a Bloom filter for storing the colors to reduce the index size. Experimental results using simulated data and real data demonstrate that our method reduces the occurrence of read-incoherent paths from 149,556 to only 2 and 5596 to 0 respectively. Moreover, the depths of coverage for the reconstructed reads are equal to those for the input reads for the simulated data, whereas the previous method decreases the depth of coverage at many positions in the source genome. Our method achieves quite a high accuracy with a comparable construction time, peak memory size, and index size to the previous method.

Abstract LB254: Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation

Abstract Sensitive and specific detection of low frequency mutations in acute myeloid leukemia (AML) is critical in research of minimal residual disease (MRD). At variant allele frequencies (VAF) below approximately 1%, PCR and sequencing errors result in prohibitive signal-to-noise ratios with next generation sequencing (NGS). Duplex Sequencing (DS) relates the original top and bottom DNA strands to make double stranded consensus sequences to greatly reduce errors. In two experiments we show further improvement of DS of AML-related genes by the use of enzymatic fragmentation (EF) and an updated gene panel that incorporates 2022 European LeukemiaNet (ELN) recommendations. In the first DS study, 29 AML-related genes (59 kb) were targeted in hybrid capture. Mutant cell line DNA was mixed into DNA from a healthy young donor to simulate MRD. Expected VAFs in mixtures were 1.0-0.003%. Samples comprised a mix of 4 insertions and deletions (indel), a mix of 15 single nucleotide variants (SNV), and 4 serial dilutions of a FLT3 ITD plus an NPM1 insertion. Pure diluent DNA was used as a negative control. Each sample was prepared in quadruplicate with mechanical fragmentation (MF) vs EF. DNA input mass was 1,500 ng per replicate, except for 50-250 ng for the FLT3/NPM1 mixes with expected VAF of 1% and 0.1%, respectively. Input masses were set to ensure >95% probability of detection of all mutations in the combined data. All targeted variants down to 0.003% VAF were detected in spike-in mixtures, with expected vs observed VAF highly correlated whether using MF or EF (R2 >0.98 for all mixtures). Duplex molecular depth was 1.2-2.0x higher with EF vs MF across input masses. Panel-wide mean duplex depth per 1,500 ng replicate was 30,376x for MF and 48,036x for EF, for combined mean depths of 121,508x for MF and 192,144x for EF. In the negative control, background mutational calls at spike-in positions were 4/2,993,429 duplex bases using MF and 0/4,780,491 duplex bases with EF, reflecting increased specificity with EF. Next, a revised panel targeting 36 AML-related genes (80 kb) was used for DS with EF. Here differing mutant cell line DNA was mixed into the same diluent from above (the negative control). This mixture harbors 27 variants with expected VAFs of 0.125-0.006% (4 indels, 21 SNVs, a FLT3 ITD and an NPM1 insertion), 17 of which (2 indels, 14 SNVs and the NPM1 insertion) span VAFs of 0.011-0.009% to establish a limit of detection of 0.01%. With 2,000 ng (+/- 10%) input, for 14 samples, panel-wide mean duplex depth was 56,528x. For variants with expected VAF ≥ 0.009%, the assay had >98% sensitivity, >96% specificity, and >98% accuracy at genomic positions of known true positive variants. In summary, DS with EF yields more data per nanogram of DNA than MF, maximizing the use of precious samples. This method exhibits extremely low background mutation signal and a low limit of detection across targets that are valuable in AML MRD research. Citation Format: Elizabeth Schmidt, Devon M. Fitzgerald, Camila Zanette, Gavin D. Meredith, Mark A. McElwain, Raul Burciaga, Kevin C. Vavra, Thomas H. Smith, Jesse J. Salk, Jake Higgins. Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB254.

High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.

Abstract 6645: A systematic approach for detection of tumor genomic alterations which alter the immune microenvironment

Abstract Objectives: Leveraging whole exome and transcriptome data from large cancer datasets, we sought to develop a computational approach to nominate candidate genomic alterations that are associated with hot or cold tumor. We anticipate this tool will identify candidate novel drug targets and biomarkers for immunotherapy in cancer. Methods: The user selects a gene of interest (GOI) as input for the pipeline. The pipeline searches TCGA (The Cancer Genome Atlas) mutation and structural variant outputs to collect hits in the gene of interest. Each sample with an alteration in the GOI is annotated with the alteration’s predicted biological function (loss-of-function or LOF vs gain-of-function or GOF) using gene set enrichment analysis and pathway analysis from RNA sequencing. After annotating by sample, the alterations themselves are scored and recorded. Next, Quantiseq and Trust4 are employed to estimate features of the immune microenvironment in both the LOF and GOF samples. These results along with cancer type distributions, genomic position maps, and oncoplots are delivered to the user for further investigation. We are creating an online application to provide these results to the public. Simultaneously, a command line tool is being developed for application to any data source. Results: We will present findings for the pipeline using positive controls including genomic alterations in ADAR, MTAP, IDH1, and CD274 (PD-L1). Preliminarily, we identified candidate GOF mutations (n=81) in PD-L1 that have inflamed tumor immune microenvironments like those of samples with known GOF structural variations in PD-L1. These samples have an overall “hot” immune microenvironment with significantly (p<.05) increased M1 macrophages, increased expression of IFN-gamma, and decreased M2 macrophages. Conclusion: Currently there are no systematic approaches to interrogate individual genomic alterations and their association with an altered immune microenvironment. Our approach is pragmatic for any gene of interest, can be applied to any database, and identifies clinically actionable targets. This approach will identify heretofore unstudied connections between genomic alterations and immune microenvironment and support personalized drug development strategies in immuno-oncology. Citation Format: Raven Vella, Emily L. Hoskins, Julie W. Reeser, Michele R. Wing, Eric Samorodnitsky, Anoosha Paruchuri, Amy Smith, Leah Stein, Thuy Dao, Zachary Risch, Sameek Roychowdhury. A systematic approach for detection of tumor genomic alterations which alter the immune microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6645.

Detection and characterization of the novel HLA-DPA1*02:66:02N allele, with a premature stop codon in exon 2.

The failure to identify HLA null alleles in bone marrow transplantation could be life-threatening because this could result in an HLA mismatch with the ability to trigger the graft-vs-host disease (GVHD) and to reduce patient's survival. In this report we describe the identification and characterization of the novel HLA-DPA1*02:66:02N allele with a non-sense codon in exon 2. This new allele was discovered in two unrelated bone marrow donors during routine HLA-typing using next-generation sequencing (NGS). DPA1*02:66:02N is homologous to DPA1*02:01:01:03 with a single nucleotide difference in exon 2, codon 50, where the replacement of C located at genomic position 3825 by T, causes the formation of a premature stop codon (TGA), resulting in a null allele. This description illustrates the benefits of HLA typing by NGS since it permits to reduce ambiguities, identify new alleles, analyze multiple HLA loci and improve transplantation outcome.

Molecular detection and phylogeography of the hypervirulent subtype of <i>Mycobacterium tuberculosis</i> Beijing 14717-15

Aim. To develop a method for detecting Mycobacterium tuberculosis strains belonging to highly lethal, hypervirulent, multidrug-resistant Beijing 14717-15-cluster to be applied to screen for M. tuberculosis retrospective collections obtained from various regions of Russia. The collection included M. tuberculosis DNA samples obtained during population studies. Spoligotyping and genotyping of 24 variable number tandem repeats loci were performed according to standard protocols. Phylogenetic analysis was based on the whole genome sequencing data. Polymorphism at the genomic position 2423040 A G, specific for the Beijing 14717-15 cluster, was detected using PCR-RFLP and HhaI restriction enzyme. The bioinformatic and phylogenetic analysis of whole genome sequencing data for 205 strains of the early ancient sublineage of the M. tuberculosis Beijing genotype showed that strains with the VNTR 14717-15 profile (according to MIRU-VNTRplus.org) and those close to it were grouped into one monophyletic cluster of related strains, which we defined as Beijing 14717-15-cluster. Among the SNPs specific for the 14717-15 cluster, we chose a functionally neutral SNP at the genomic position 2423040 A G (Rv2161c Val33Ala) and developed a method for its detection in the PCR-RFLP format. Next, we applied this method to screening DNA collections from the regions of the European and Asian parts of Russia and Asian countries. The data demonstrate the presence of strains of the Beijing 14717-15 cluster only in the Asian region of Russia peaking in Buryatia (18%). We have developed a method for detection of the hypervirulent and highly lethal genetic cluster M. tuberculosis Beijing 14717-15 based on detection of the specific mutation in the Rv2161c gene using PCR followed by HhaI restriction of the PCR product and agarose gel electrophoresis to discriminate between wild-type and mutant alleles. The advantages of the proposed method are the speed, simple and unambiguous data interpretation, opportunity to detect contamination and mixed infection, suitability for efficient analysis of large collections of M. tuberculosis strains.

Reliable genotyping of recombinant genomes using a robust hidden Markov model.

Meiotic recombination is an essential mechanism during sexual reproduction and includes the exchange of chromosome segments between homologous chromosomes. New allelic combinations are transmitted to the new generation, introducing novel genetic variation in the offspring genomes. With the improvement of high-throughput whole-genome sequencing technologies, large numbers of recombinant individuals can now be sequenced with low sequencing depth at low costs, necessitating computational methods for reconstructing their haplotypes. The main challenge is the uncertainty in haplotype calling that arises from the low information content of a single genomic position. Straightforward sliding window-based approaches are difficult to tune and fail to place recombination breakpoints precisely. Hidden Markov model (HMM)-based approaches, on the other hand, tend to over-segment the genome. Here, we present RTIGER, an HMM-based model that exploits in a mathematically precise way the fact that true chromosome segments typically have a certain minimum length. We further separate the task of identifying the correct haplotype sequence from the accurate placement of haplotype borders, thereby maximizing the accuracy of border positions. By comparing segmentations based on simulated data with known underlying haplotypes, we highlight the reasons for RTIGER outperforming traditional segmentation approaches. We then analyze the meiotic recombination pattern of segregants of 2 Arabidopsis (Arabidopsis thaliana) accessions and a previously described hyper-recombining mutant. RTIGER is available as an R package with an efficient Julia implementation of the core algorithm.

Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.

Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9's application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichiacoli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR-Cas experiments in bacteria.

Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems-the only DNA transposons currently employed in clinical trials-during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

Genomic Analysis Reveals New Integrative Conjugal Elements and Transposons in GBS Conferring Antimicrobial Resistance.

Streptococcus agalactiae or group B streptococcus (GBS) is a leading cause of neonatal sepsis and increasingly found as an invasive pathogen in older patient populations. Beta-lactam antibiotics remain the most effective therapeutic with resistance rarely reported, while the majority of GBS isolates carry the tetracycline resistance gene tet(M) in fixed genomic positions amongst five predominant clonal clades. In the UK, GBS resistance to clindamycin and erythromycin has increased from 3% in 1991 to 11.9% (clindamycin) and 20.2% (erythromycin), as reported in this study. Here, a systematic investigation of antimicrobial resistance genomic content sought to fully characterise the associated mobile genetic elements within phenotypically resistant GBS isolates from 193 invasive and non-invasive infections of UK adult patients collected during 2014 and 2015. Resistance to erythromycin and clindamycin was mediated by erm(A) (16/193, 8.2%), erm(B) (16/193, 8.2%), mef(A)/msr(D) (10/193, 5.1%), lsa(C) (3/193, 1.5%), lnu(C) (1/193, 0.5%), and erm(T) (1/193, 0.5%) genes. The integrative conjugative elements (ICEs) carrying these genes were occasionally found in combination with high gentamicin resistance mediating genes aac(6')-aph(2″), aminoglycoside resistance genes (ant(6-Ia), aph(3'-III), and/or aad(E)), alternative tetracycline resistance genes (tet(O) and tet(S)), and/or chloramphenicol resistance gene cat(Q), mediating resistance to multiple classes of antibiotics. This study provides evidence of the retention of previously reported ICESag37 (n = 4), ICESag236 (n = 2), and ICESpy009 (n = 3), as well as the definition of sixteen novel ICEs and three novel transposons within the GBS lineage, with no evidence of horizontal transfer.