Plasmid Integration Research Articles

Development and Properties of Francisella tularensis Subsp. holarctica 15 NIIEG Vaccine Strain without the recD Gene.

The genomic analysis of all subspecies F. tularensis, as found in Gen Bank NCBI, reveals the presence of genes encoding proteins like to the multifunctional RecBCD enzyme complex in E. coli and other bacteria. To date, the role of the recD gene in F. tularensis, which encodes the alpha chain of exonuclease V, in DNA metabolism processes, has not been studied either in vitro or in vivo. F. tularensis subsp. holarctica 15 NIIEG, a vaccine strain, served as the basis to create the F. tularensis 15D strain with recD deletion. The lack of the recD gene suppresses the integration of suicide plasmids with F. tularensis genome fragments into the chromosome. The modified strain showed reduced growth in vitro and in vivo. This study shows that such deletion significantly reduces the virulence of the strain in BALB/c mice.

New<i> Bacillus subtilis</i> vector, pSSβ, as genetic tool for site-specific integration and excision of cloned DNA, and prophage elimination

Site-specific recombination (SSR) systems are employed in many genetic mobile elements, including temperate phages, for their integration and excision. Recently, they have also been used as tools for applications in fields ranging from basic to synthetic biology. SPβ is a temperate phage of the Siphoviridae family found in the laboratory standard Bacillus subtilis strain 168. SPβ encodes a serine-type recombinase, SprA, and recombination directionality factor (RDF), SprB. SprA catalyzes recombination between the attachment site of the phage, attP, and that of the host, attB, to integrate phage genome into the attB site of the host genome and generate attL and attR at both ends of the prophage genome. SprB works in conjunction with SprA and switches from attB/attP to attL/R recombination, which leads to excision of the prophage. In the present study, we took advantage of this highly efficient recombination system to develop a site-specific integration and excision plasmid vector, named pSSβ. It was constructed using pUC plasmid and the SSR system components, attP, sprA and sprB of SPβ. pSSβ was integrated into the attB site with a significantly high efficiency, and the resulting pSSβ integrated strain also easily eliminated pSSβ itself from the host genome by the induction of SprB expression with xylose. This report presents two applications using pSSβ that are particularly suitable for gene complementation experiments and for a curing system of SPβ prophage, that may serve as a model system for the removal of prophages in other bacteria.

Double Crossover Approach to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC7120 (hereafter Anabaena 7120) is a nitrogen-fixing, filamentous cyanobacterium. Given its diverse metabolism, it serves as an excellent model organism, particularly for studying cell differentiation, nitrogen fixation, photosynthesis, production of high-value chemicals, and synthetic biology. Gene knockout is a common approach to assess the function of gene products through assessing phenotypic loss of function. In the method described here, a double crossover approach is used to inactivate a target gene or target genes in Anabaena 7120. This method involves replicating the gene(s) from the wild-type genomic DNA and inserting them into an integrative plasmid vector. An internal portion of the genes may be removed and replaced with a GFP-Spectinomycin (gfp-sp) cassette. The plasmid is then introduced into Anabaena 7120 where a double crossover event occurs between the wild-type chromosome and the cargo plasmid, effectively replacing the wild-type gene with the disrupted gene from the plasmid. The gfp-sp cassette combined with the sacB gene serve as positive selection to identify double crossover mutants (Caiand Wolk(1990), 172(6):3138-3145, J.Bacteriol). Finally, the functional genes are cloned into another replicating plasmid vector to produce a cargo plasmid, which is conjugatively introduced into the mutant for a complementation test. By comparing the phenotypes among the wild-type, mutant, and complement, one should see a loss of function in the mutant which is recovered in the complement, thereby defining the function of the target gene. The double crossover approach described here for Anabaena PCC 7120 may be broadly applicable to the study of gene function in cyanobacteria and other prokaryotic organisms.

Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system.

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and that biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV both in vitro and in fission yeast. We introduced SynPCB2.1, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB2.1. These tools not only enable the easy use of multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms. This article has an associated First Person interview with the first author of the paper.

Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing.

The targeted deletion, replacement, integration, or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences including uncontrolled indel mixtures and chromosomal abnormalities. Here, we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the PE-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and may synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces.

Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces .

Frequency, composition and mobility of Escherichia coli-derived transposable elements in holdings of plasmid repositories.

SummaryBy providing the scientific community with uniform and standardized resources of consistent quality, plasmid repositories play an important role in enabling scientific reproducibility. Plasmids containing insertion sequence elements (IS elements) represent a challenge from this perspective, as they can change the plasmid structure and function. In this study, we conducted a systematic analysis of a subset of plasmid stocks distributed by plasmid repositories (The Arabidopsis Biological Resource Center and Addgene) which carry unintended integrations of bacterial mobile genetic elements. The integration of insertion sequences was most often found in, but not limited to, pBR322‐derived vectors, and did not affect the function of the specific plasmids. In certain cases, the entire stock was affected, but the majority of the stocks tested contained a mixture of the wild‐type and the mutated plasmids, suggesting that the acquisition of IS elements likely occurred after the plasmids were acquired by the repositories. However, comparison of the sequencing results of the original samples revealed that some plasmids already carried insertion mutations at the time of donation. While an extensive BLAST analysis of 47 877 plasmids sequenced from the Addgene repository uncovered IS elements in only 1.12%, suggesting that IS contamination is not widespread, further tests showed that plasmid integration of IS elements can propagate in conventional Escherichia coli hosts over a few tens of generations. Use of IS‐free E. coli hosts prevented the emergence of IS insertions as well as that of small indels, suggesting that the use of IS‐free hosts by donors and repositories could help limit unexpected and unwanted IS integrations into plasmids.

A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid.

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and was confirmed using whole-genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Alanine-scanning mutagenesis of protein mannosyl-transferase from Streptomyces coelicolor reveals strong activity-stability correlation.

In Actinobacteria , protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation has an important role in cell envelope physiology. In S. coelicolor, defective Pmt leads to increased susceptibility to cell wall-targeting antibiotics, including vancomycin and β-lactams, and resistance to phage ϕC31. The aim of this study was to gain a deeper understanding of the structure and function of S. coelicolor Pmt. Sequence alignments and structural bioinformatics were used to identify target sites for an alanine-scanning mutagenesis study. Mutant alleles were introduced into pmt-deficient S. coelicolor strains using an integrative plasmid and scored for their ability to complement phage resistance and antibiotic hypersusceptibility phenotypes. Twenty-three highly conserved Pmt residues were each substituted for alanine. Six mutant alleles failed to complement the pmt ▬ strains in either assay. Mapping the six corresponding residues onto a homology model of the three-dimensional structure of Pmt, indicated that five are positioned close to the predicted catalytic DE motif. Further mutagenesis to produce more conservative substitutions at these six residues produced Pmts that invariably failed to complement the DT1025 pmt ▬ strain, indicating that strict residue conservation was necessary to preserve function. Cell fractionation and Western blotting of strains with the non-complementing pmt alleles revealed undetectable levels of the enzyme in either the membrane fractions or whole cell lysates. Meanwhile for all of the strains that complemented the antibiotic hypersusceptibility and phage resistance phenotypes, Pmt was readily detected in the membrane fraction. These data indicate a tight correlation between the activity of Pmt and its stability or ability to localize to the membrane.

Encapsulation of Large-Size Plasmids in PLGA Nanoparticles for Gene Editing: Comparison of Three Different Synthesis Methods.

The development of new gene-editing technologies has fostered the need for efficient and safe vectors capable of encapsulating large nucleic acids. In this work we evaluate the synthesis of large-size plasmid-loaded PLGA nanoparticles by double emulsion (considering batch ultrasound and microfluidics-assisted methodologies) and magnetic stirring-based nanoprecipitation synthesis methods. For this purpose, we characterized the nanoparticles and compared the results between the different synthesis processes in terms of encapsulation efficiency, morphology, particle size, polydispersity, zeta potential and structural integrity of loaded pDNA. Our results demonstrate particular sensibility of large pDNA for shear and mechanical stress degradation during double emulsion, the nanoprecipitation method being the only one that preserved plasmid integrity. However, plasmid-loaded PLGA nanoparticles synthesized by nanoprecipitation did not show cell expression in vitro, possibly due to the slow release profile observed in our experimental conditions. Strong electrostatic interactions between the large plasmid and the cationic PLGA used for this synthesis may underlie this release kinetics. Overall, none of the methods evaluated satisfied all the requirements for an efficient non-viral vector when applied to large-size plasmid encapsulation. Further optimization or alternative synthesis methods are thus in current need to adapt PLGA nanoparticles as delivery vectors for gene editing therapeutic technologies.

A Regulator Based "Semi-Targeted" Approach to Activate Silent Biosynthetic Gene Clusters.

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Analysis of the efficiency factors of electrotransformation of Bacillus subtilis to inactivate the aroK gene by the method of homologous recombination

A hyper-osmotic electrotransformation method was developed for strain Bacillus subtilis. Sorbitol and mannitol are included in the hyper-osmotic electroporation medium and recovery medium. In this study, the hyper-osmotic electroporation method was optimised to increase the transformation efficiency of B. subtilis strain 5434 (non-transformable by chemical methods) by 430 fold, with a maximum value of 8.6 ⋅ 105 CFU/µg of integrative plasmid DNA. With the electroporation setted 25 µF, 23 kV/cm, 200 Ω, the method was optimised as follows: a) the OD600 value of the bacterial culture solution was increased to about 1.2, which significantly enhanced survival of bacteria and quantity of viable B.subtilis strain 5434 cells after electroporation; b) the elution frequency of washing solution (hyper-osmotic electroporation medium) for complement cells was increased from 3 to 5 times, resulted in significantly reducing the conductivity of the hyper-osmotic electoporation medium with competent cells (electrocompetent cultue), and effectively extending the pulse time under the same electric field strength; c) quantity of integrative plasmid DNA added to hyper-osmotic electrocompetent culture was optimised. These results indicate that increasing the number of viable B. subtilis strain 5434 cells and reducing the number of metal ions in the electroporation solution mix (integrative plasmid DNA, competent cells of B. subtilis strain 5434, electroporation medium) are useful approach to improve transfomation efficiency of B. subtilis strain 5434. Concentration of shikimic acid in the fermentation medium was quantified by high performance liquid chromatography. Quantification of shikimic acid revealed that B. subtilis strain 5434p4SA produced 403.98 ± 9.1 µg/mL of shikimic acid.

The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis

BackgroundAnimal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour.ResultsNo off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations.ConclusionsWe found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

Genomic portrait of community-associated methicillin-resistant Staphylococcus aureus ST772-SCCmec V lineage from India

BackgroundSignificant changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognised with the emergence of community-associated methicillin-resistant Staphylococcus aureus. However, studies on the molecular epidemiology and the genomic investigation of MRSA are limited in India. AimThe study was aims to understand the molecular epidemiology of MRSA causing bloodstream infection and also to investigate the origin and evolution of ST772 S. aureus isolated from India. MethodsA total of 233 non-repetitive MRSA isolates were screened for the presence staphylococcal cassette chromosome (SCCmec) types, multi-locus sequence types (MLST) and staphylococcal protein A (spa) types. Whole genome sequence data of ST772-SCCmec V (n = 32) isolates were generated and comparative analysis was performed. ResultsMLST analysis revealed ten different clonal complexes and three singletons. ST772 (27%), ST22 (19%) and ST239 (16%) were the predominant MRSA genotypes in causing bloodstream infection. The spa types were highly diverse. Phylogenetic analysis revealed that nearly three-fourth of the Indian STT72-SCCmec V isolates belongs to dominant (ST772-A2) and emerging subgroups (ST772-A3). A pattern of increasing antimicrobial resistance was noticed in the dominant and emerging subgroups. An integrated resistance plasmid encoding resistance clusters for beta-lactam (blaZ), macrolides (mphC, msrA), and aminoglycoside resistance (aphA-III, sat-4, aadE) was identified in all isolates, except four basal strains. ST772-SCCmec V was emerged on the Indian subcontinent in 1964 and diverged into a dominant subgroup in 1991. Furthermore, the expansion is likely to be associated with the acqusition of mobile genetic elements such as integrated resistance plasmid and SCCmec V (5C2) as well as the fixation of double serine mutation (S84L, S80Y) in the quinolone resistance determining region. ConclusionsST772 S. aureus have consistent virulence and resistance determinants which may results in successful survival in both community and hospital settings.

Retrospective assessment of clonal origin of cell lines.

Cell lines used for the manufacture of recombinant proteins are expected to arise from a single cell as a control strategy to limit variability and ensure consistent protein production. Health authorities require a minimum of two rounds of limiting dilution cloning or its equivalent to meet the requirement of single cell origin. However, many legacy cell lines may not have been generated with process meeting this criteria potentially impeding the path to commercialization. A general monoclonality assessment strategy was developed based on using the site of plasmid integration for a cell's identity. By comparing the identities of subclones from a master cell bank (MCB) to each other and that of the MCB, a probability of monoclonality was established. Two technologies were used for cell identity, Southern blot and a PCR assay based on plasmid-genome junction sequences identified by splinkerette PCR. Southern blot analysis revealed that subclones may have banding patterns that differ from each other and yet indicate monoclonal origin. Splinkerette PCR identifies cellular sequence flanking the point(s) of plasmid integration. The two assays together provide complimentary data for cell identity that enables proper monoclonality assessment and establishes that the three legacy cell lines investigated are all of clonal origin.

Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens.

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

Chlorine disinfection facilitates natural transformation through ROS-mediated oxidative stress

The bacterial infection that involves antimicrobial resistance is a rising global threat to public health. Chlorine-based water disinfection processes can inactivate antibiotic resistant bacteria. However, at the same time, these processes may cause the release of antibiotic resistance genes into the water as free DNA, and consequently increase the risk to disseminate antibiotic resistance via natural transformation. Presently, little is known about the contribution of residual chlorine affecting the transformation of extracellular antibiotic resistance genes (ARGs). This study investigates whether chloramine and free chlorine promote the transformation of ARGs and how this may occur. We reveal that both chloramine and free chlorine, at practically relevant concentrations, significantly stimulated the transformation of plasmid-encoded ARGs by the recipient Acinetobacter baylyi ADP1, by up to a 10-fold increase. The underlying mechanisms underpinning the increased transformations were revealed. Disinfectant exposure induced a series of cell responses, including increased levels of reactive oxygen species (ROS), bacterial membrane damage, ROS-mediated DNA damage, and increased stress response. These effects thus culminated in the enhanced transformation of ARGs. This promoted transformation was observed when exposing disinfectant-pretreated A. baylyi to free plasmid. In contrast, after pretreating free plasmid with disinfectants, the transformation of ARGs decreased due to the damage of plasmid integrity. These findings provide important insight on the roles of disinfectants affecting the horizontal transfer of ARGs, which could be crucial in the management of antibiotic resistance in our water systems.

Arpin Regulates Migration Persistence by Interacting with Both Tankyrases and the Arp2/3 Complex.

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid–liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the ARPIN locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.

An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.

Self-Cutting and Integrating CRISPR Plasmids Enable Targeted Genomic Integration of Genetic Payloads for Rapid Cell Engineering.

Since observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited insertion or deletion formation. Interestingly, including longer homology arms (HAs; 500 bp) in varying orientations only modestly increased knock-in efficiency (by around twofold). Using a SCIP-payload design (SCIPpay) that liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs of concept, we used SCIPpay to insert (1) a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a cell line that reports T-cell activation, and (2) a chimeric antigen receptor gene into the TRAC locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines, and we suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.