Search

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.

CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

Objective The composition of the healthy human adult gut microbiome is relatively stable over prolonged periods, and representatives of the most highly abundant and prevalent species have been cultured and described. However, microbial abundances can change on perturbations, such as antibiotics intake, enabling the identification and characterisation of otherwise low abundant species. Design Analysing gut microbial time-series data, we used shotgun metagenomics to create strain level taxonomic and functional profiles. Community dynamics were modelled postintervention with a focus on conditionally rare taxa and previously unknown bacteria. Results In response to a commonly prescribed cephalosporin (ceftriaxone), we observe a strong compositional shift in one subject, in which a previously unknown species, U Borkfalki ceftriaxensis, was identified, blooming to 92% relative abundance. The genome assembly reveals that this species (1) belongs to a so far undescribed order of Firmicutes, (2) is ubiquitously present at low abundances in at least one third of adults, (3) is opportunistically growing, being ecologically similar to typical probiotic species and (4) is stably associated to healthy hosts as determined by single nucleotide variation analysis. It was the first coloniser after the antibiotic intervention that led to a long-lasting microbial community shift and likely permanent loss of nine commensals. Conclusion The bloom of U B. ceftriaxensis and a subsequent one of Parabacteroides distasonis demonstrate the existence of monodominance community states in the gut. Our study points to an undiscovered wealth of low abundant but common taxa in the human gut and calls for more highly resolved longitudinal studies, in particular on ecosystem perturbations., Gut, 68 (10), ISSN:1468-3288, ISSN:0017-5749

Pre-capture multiplexing is a cost-effective and efficient way for targeted next-generation sequencing. To allow high-throughput pre-capture pooling without compromising the sequencing quality, we developed a series of reagents, including 384+ plex customizable adaptors, TRUE universal blockers, and a fast and cost-effective hybrid capture solution using semiconductor DNA synthesis technology. Our data shows our adaptors have an extremely lost cross-contamination rate, which can minimize the index hopping during pre-capture pooling. The TRUE universal blockers using unique chemistry can bind to adapters regardless of the index length, simplify the workflow and still yield consistent capture efficiency during sample multiplexing. Also, the GenFisher capture probes, produced by semiconductor DNA synthesis technology, can deliver high capture efficiency after pre-capture pooling, with the potential of evolution via the test-learn cycle.

Hybrid capture is a popular and well-accepted method to select targeted genomic regions for subsequent next generation sequencing (NGS). Despite its popularity in research and clinical diagnostics, hybrid capture NGS is now facing the challenge by the high throughput demands generated by Novaseq, Illumina's flagship platform that occupies the research and industry market rapidly in recent years. Novaseq create tetrabytes of sequencing data per run, allowing unprecedented level of sample multiplexing, especially for targeted NGS. Conventional blockers, whose sample index-matching region uses either degenerate bases or universal pairing bases, still need to match the length of the sample index for each individual library. When using NovaSeq, scientists need to combine huge number of samples using different types of index system in each single run. However, matching different hybridization blockers with corresponding index system is time consuming and error-prone. Here we introduce a novel design and chemistry to hybrid capture blockers to maximized the pooling capacity of hybrid capture to match the increasing need of sample throughput and multiplexing. Our new hybrid capture blockers are not only an essential component in the target enrichment workflow to block the permissive interactions between library adapters and target probes, which may lower the capturing efficiency and generate excessive data from the off-target sequence, but also significantly promotes the capturing capacity and efficiency, and simplifies the workflow. Our new blocker, GenFisher Universal Blockers, using our proprietary design and chemistry, create a novel solution to the conjugation between sample indexes on libraries and blockers, enabling blockers bind adapters regardless to the index length and sequence. In our experiment, NGS libraries are created with a commercially available kit and TruSeq style adapters that contain sample indexes with 3 different lengths: 6nt, 8nt and 10nt. Hybrid captures were then carried out with 8,000-probe GenFisher hybridization panel and kit and Universal Blockers for each individual libraries and the combined library with different indexes. Sequencing data showed that the on-target base percentage was consistently at 30% on-target rate. We also compared GenFisher Universal Blockers with the blockers from two other industrial leading providers, which are also claimed to be ‘universal'. Our data showed that GenFisher Universal Blockers was the only truly universal solution that worked with all 3 index lengths unbiasedly while the other two are still index length-sensitive and struggled with 10nt indexes library capturing (on-target ~50%, compared to Citation Format: Jianpeng Wang, Long Fan, Lumeng Ye, Jiashi Wang, Irene Song, Cedric Wu. Enhanced target capturing throughput and simplified workflow via innovative chemistry on hybrid capture blockers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1352.

Marine sponges (phylum Porifera) are well known to harbour a complex and diverse bacterial community. Some of these sponge-associated bacteria have been shown to be the real producers of secondary metabolites with a wide range of activities from antimicrobials to anticancer agents. Previously, we revealed that the strain Pseudomonas fluorescens H41 isolated from the sponge Haliclona sp. (collected at the coast of Rio de Janeiro, Brazil) showed a strong antimicrobial activity against clinical and marine bacteria. Thus, in this study the genes involved in the antimicrobial activity of P. fluorescens H41 were identified. To this end, a library of mutants was generated via miniTnphoA3 transposon mutagenesis and the resulting clones were characterized for their antimicrobial activity. It was demonstrated that genes involved in the biosynthesis of the pyoverdine siderophore are related to the inhibitory activity of P. fluorescens H41. Therefore, this strain might play an important role in the biocontrol of the host sponge.

Mupirocin is a polyketide antibiotic with broad antibacterial activity. It was isolated and characterized about 40 years ago from Pseudomonas fluorescens NCIMB 10586. To study the phylogenetic distribution of mupirocin producing strains in the genus Pseudomonas a large collection of Pseudomonas strains of worldwide origin, consisting of 117 Pseudomonas type strains and 461 strains isolated from different biological origins, was screened by PCR for the mmpD gene of the mupirocin gene cluster. Five mmpD + strains from different geographic and biological origin were identified. They all produced mupirocin and were strongly antagonistic against Staphylococcus aureus . Phylogenetic analysis showed that mupirocin production is limited to a single species. Inactivation of mupirocin production leads to complete loss of in vitro antagonism against S. aureus , except on certain iron-reduced media where the siderophore pyoverdine is responsible for the in vitro antagonism of a mupirocin-negative mutant. In addition to mupirocin some of the strains produced lipopeptides of the massetolide group. These lipopeptides do not play a role in the observed in vitro antagonism of the mupirocin producing strains against S. aureus .

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as -L-Asp-L-Ala-D-AOHOrn-L-Thr-Gly-c[L-Thr(O-)-L-Hse-D-Hya-L-Ser-L-Orn-L-Hse-L-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C65H93N17O32, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.

Pseudomonas aeruginosa is an important opportunistic pathogen. Strain FRD1 is a mucoid isolate from the sputum of a cystic fibrosis patient. It has been widely studied and has many different phenotypes compared to nonmucoid strains. Here, we present the draft genome sequence of P. aeruginosa strain FRD1 to gain insight into mucoid isolates.

Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors. PMID: 25369289 [PubMed - in process] PMCID: PMC4219678 Free PMC Article

Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors.

An antibiotic substance isolated from Pseudomonas fluorescens strain G308 was earlier assigned the structure of N-mercapto-4-formylcarbostyril, but computational predictions of the 1H and 13C NMR magnetic shielding tensors show this structure to be incompatible with the published spectroscopic data. The same is true for six quinoline derivatives related to N-mercapto-4-formylcarbostyril by permutation of the O and S atoms. In contrast, 2-(2-hydroxyphenyl)thiazole-4-carbaldehyde [aeruginaldehyde], isolated from Pseudomonas protegens Pf-5, together with the reduced derivative aeruginol, displays spectroscopic data identical with those of the alleged carbostyril derivative. In addition, the published 1H and 13C NMR data are in agreement with those calculated for aeruginaldehyde. We propose that aeruginaldehyde and aeruginol originate from the non-ribosomal peptide synthetase enzymes involved in the siderophores enantio-pyochelin (or pyochelin) biosynthetic pathways.

All fluorescent pseudomonads (Pseudomonas aeruginosa, P. putida, P. fluorescens, P. syringae and others) are known to produce the high-affinity peptidic yellow-green fluorescent siderophore pyoverdine. These siderophores have peptide chains that are quite diverse and more than 50 pyoverdine structures have been elucidated. In the majority of the cases, a Pseudomonas species is also able to produce a second siderophore of lower affinity for iron. Pseudomonas fluorescens ATCC 17400 has been shown to produce a unique second siderophore, (thio)quinolobactin, which has an antimicrobial activity against the phytopathogenic Oomycete Pythium debaryanum. We show that this strain has the capacity to utilize 16 different pyoverdines, suggesting the presence of several ferripyoverdine receptors. Analysis of the draft genome of P. fluorescens ATCC 17400 confirmed the presence of 55 TonB-dependent receptors, the largest so far for Pseudomonas, among which 15 are predicted to be ferripyoverdine receptors (Fpv). Phylogenetic analysis revealed the presence of two different clades containing ferripyoverdine receptors, with sequences similar to the P. aeruginosa type II FpvA forming a separate cluster. Among the other receptors we confirmed the presence of the QbsI (thio)quinolobactin receptor, an ferri-achromobactin and an ornicorrugatin receptor, several catecholate and four putative heme receptors. Twenty five of the receptors genes were found to be associated with genes encoding extracytoplasmic sigma factors (ECF σ) and transmembrane anti-σ sensors.

Chronic Pseudomonas aeruginosa infections are the main cause of morbidity among patients with cystic fibrosis (CF) due to persistent lung inflammation caused by interaction between this bacterium and the immune system. Longitudinal studies of clonally related isolates of a dominant CF clone have indicated that genome reduction frequently occurs during adaptation of P. aeruginosa in the CF lung. In this study, we have evaluated the P. aeruginosa population structure of patients attending the Universitair Ziekenhuis Brussel (UZ Brussel) CF reference center using a combination of genotyping methods. Although the UZ Brussel P. aeruginosa CF population is characterized by the absence of a dominant CF clone, some potential interpatient transmissions could be detected. Interestingly, one of these clones showed deletion of the alternative type I ferripyoverdine receptor gene fpvB . Furthermore, we found that several other TonB-dependent receptors are deleted as well. The genome of one potentially transmissible CF clone was sequenced, revealing large deleted regions including all type III secretion system genes and several virulence genes. Remarkably, a large number of deleted genes are shared between the P. aeruginosa CF clone described in this study and isolates belonging to the dominant Copenhagen CF DK2 clone, suggesting parallel evolution.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to definethe receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain ?-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as -l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C65H93N17O32, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.

The skin secretion of many amphibians contains an arsenal of bioactive molecules, including hormone-like peptides (HLPs) acting as defense toxins against predators, and antimicrobial peptides (AMPs) providing protection against infectious microorganisms. Several amphibian taxa seem to have independently acquired the genes to produce skin-secreted peptide arsenals, but it remains unknown how these originated from a non-defensive ancestral gene and evolved diverse defense functions against predators and pathogens. We conducted transcriptome, genome, peptidome and phylogenetic analyses to chart the full gene repertoire underlying the defense peptide arsenal of the frog Silurana tropicalis and reconstruct its evolutionary history. Our study uncovers a cluster of 13 transcriptionally active genes, together encoding up to 19 peptides, including diverse HLP homologues and AMPs. This gene cluster arose from a duplicated gastrointestinal hormone gene that attained a HLP-like defense function after major remodeling of its promoter region. Instead, new defense functions, including antimicrobial activity, arose by mutation of the precursor proteins, resulting in the proteolytic processing of secondary peptides alongside the original ones. Although gene duplication did not trigger functional innovation, it may have subsequently facilitated the convergent loss of the original function in multiple gene lineages (subfunctionalization), completing their transformation from HLP gene to AMP gene. The processing of multiple peptides from a single precursor entails a mechanism through which peptide-encoding genes may establish new functions without the need for gene duplication to avoid adaptive conflicts with older ones., Author Summary Many amphibians defend themselves against predation and infections by secreting a mixture of gene-encoded toxins and antimicrobials. How does such an integrated defense weapon arise and how does it diversify to gain distinct antipredatory and antimicrobial functions? We took advantage of the availability of a sequenced genome for the African clawed frog Silurana tropicalis to provide the first comprehensive overview of an amphibian peptide defense arsenal, from its underlying genes to its bioactive components. A reconstruction of the evolutionary history of this gene repertoire allows us to elucidate the timing and mode of evolution of distinct defense functions. Our study shows that the basal transition from a gastrointestinal hormone function to a skin-secretory defense function was accompanied by major restructuring of regulatory sequences in the ancestral gene. Instead, subsequently diversifying defense genes underwent functional shifts by entering a bifunctional stage (by cleavage of two distinct defense peptides from a single precursor protein) and occasionally losing the original defense function (by loss of the original defense peptide). This pattern provides an evolutionary explanation for the processing of structurally or functionally unrelated toxins from the same or closely related precursor proteins in other poisonous and venomous animals.

An antibiotic substance isolated from Pseudomonas fluorescens strain G308 was earlier assigned the structure of N-mercapto-4-formylcarbostyril, but computational predictions of the 1H and 13C NMR magnetic shielding tensors show this structure to be incompatible with the published spectroscopic data. The same is true for six quinoline derivatives related to N-mercapto-4-formylcarbostyril by permutation of the O and S atoms. In contrast, 2-(2-hydroxyphenyl)thiazole-4-carbaldehyde [aeruginaldehyde], isolated from Pseudomonas protegens Pf-5, together with the reduced derivative aeruginol, displays spectroscopic data identical with those of the alleged carbostyril derivative. In addition, the published 1H and 13C NMR data are in agreement with those calculated for aeruginaldehyde. We propose that aeruginaldehyde and aeruginol originate from the non-ribosomal peptide synthetase enzymes involved in the siderophores enantio-pyochelin (or pyochelin) biosynthetic pathways.