Excision Of Cassettes Research Articles

Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing

CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from "scarless editing by element deletion"), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.

Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs.

Cassette recombination dynamics within chromosomal integrons are regulated by toxin-antitoxin systems.

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here, we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.

SLICER: A Seamless Gene Deletion Method for Deinococcus radiodurans.

Deinococcus radiodurans' high resistance to various stressors combined with its ability to utilize sustainable carbon sources makes it an attractive bacterial chassis for synthetic biology and industrial bioproduction. However, to fully harness the capabilities of this microbe, further strain engineering and tool development are required. Methods for creating seamless genome modifications are an essential part of the microbial genetic toolkit to enable strain engineering. Here, we report the development of the SLICER method, which can be used to create seamless gene deletions in D. radiodurans. This process involves (a) integration of a seamless deletion cassette replacing a target gene, (b) introduction of the pSLICER plasmid to mediate cassette excision by I-SceI endonuclease cleavage and homologous recombination, and (c) curing of the helper plasmid. We demonstrate the utility of SLICER for creating multiple gene deletions in D. radiodurans by sequentially targeting 5 putative restriction-modification system genes, recycling the same selective and screening markers for each subsequent deletion. While we observed no significant increase in transformation efficiency for most of the knockout strains, we demonstrated SLICER as a promising method to create a fully restriction-minus strain to expand the synthetic biology applications of D. radiodurans, including its potential as an invivo DNA assembly platform.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

SummaryWe describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

Differences in Integron Cassette Excision Dynamics Shape a Trade-Off between Evolvability and Genetic Capacitance.

ABSTRACTIntegrons ensure a rapid and “on demand” response to environmental stresses driving bacterial adaptation. They are able to capture, store, and reorder functional gene cassettes due to site-specific recombination catalyzed by their integrase. Integrons can be either sedentary and chromosomally located or mobile when they are associated with transposons and plasmids. They are respectively called sedentary chromosomal integrons (SCIs) and mobile integrons (MIs). MIs are key players in the dissemination of antibiotic resistance genes. Here, we used in silico and in vivo approaches to study cassette excision dynamics in MIs and SCIs. We show that the orientation of cassette arrays relative to replication influences attC site folding and cassette excision by placing the recombinogenic strands of attC sites on either the leading or lagging strand template. We also demonstrate that stability of attC sites and their propensity to form recombinogenic structures also regulate cassette excision. We observe that cassette excision dynamics driven by these factors differ between MIs and SCIs. Cassettes with high excision rates are more commonly found on MIs, which favors their dissemination relative to SCIs. This is especially true for SCIs carried in the Vibrio genus, where maintenance of large cassette arrays and vertical transmission are crucial to serve as a reservoir of adaptive functions. These results expand the repertoire of known processes regulating integron recombination that were previously established and demonstrate that, in terms of cassette dynamics, a subtle trade-off between evolvability and genetic capacitance has been established in bacteria.

Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors

Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60–70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.

P035 Adipocyte derived stem cells favour healing in a new minipig model of cutaneous radiation syndrome through reepithelialisation and neoangiogenesis

Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60–70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.

The role of adipose-derived stem cells engineered with the persistently expressing hybrid baculovirus in the healing of massive bone defects

Massive segmental defects arising from trauma or tumor resection remain a challenging clinical problem. To repair large, segmental bone defects using adipose-derived stem cells (ASCs) which alone cannot heal massive defects, we hypothesized that sustained expression of factors promoting osteogenesis (BMP2) and angiogenesis (VEGF) provides continuous stimuli to augment the healing. Baculovirus is a vector for gene delivery into stem cells, but it only mediates transient expression. Therefore we developed a dual system whereby one baculovirus expressed FLP recombinase (BacFLP) while the other hybrid baculovirus harbored an Frt-flanking transgene cassette. Within the ASCs transduced with BacFLP and the hybrid baculovirus, the transduction efficiency reached 98% and the FLP/Frt-mediated recombination efficiency approached 46%, leading to cassette excision off the baculovirus genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days. ASCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF (S group) only healed the critical-size (10mm) segmental femoral bone defects in 40% of New Zealand White rabbits at 12 weeks post-implantation, whereas ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-size defects in 12 out of 12 animals in 8 weeks. Compared with the S group, the L group not only accelerated the healing, but also ameliorated the bone quality (metabolism, volume, density, mechanical properties) and angiogenesis, thereby attesting our hypothesis that persistent BMP2/VEGF expression is essential. Use of ASCs engineered by the hybrid BV vector thus holds promise to treat massive segmental defects necessitating sustained stimuli.

Effect of attC structure on cassette excision by integron integrases

BackgroundIntegrons are genetic elements able to integrate and disseminate genes as cassettes by a site-specific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. Integron integrases (IntIs) bind specifically to the bottom strand of attC sites. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases. These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. Thus, it is important to better understand interactions between IntIs and their substrates.ResultsWe compared the ability of five IntIs to carry out excision of several cassettes flanked by different attC sites. The results showed that for most cassettes, IntI1 was the most active integrase. However, IntI2*179E and SonIntIA could easily excise cassettes containing the attCdfrA1 site located upstream, whereas IntI1 and IntI3 had only a weak excision activity for these cassettes. Analysis of the secondary structure adopted by the bottom strand of attCdfrA1 has shown that the identity of the extrahelical bases and the distance between them (A-N7-8-C) differ from those of attCs contained in the cassettes most easily excisable by IntI1 (T-N6-G). We used the attCdfrA1 site upstream of the sat2 gene cassette as a template and varied the identity and spacing between the extrahelical bases in order to determine how these modifications influence the ability of IntI1, IntI2*179E, IntI3 and SonIntIA to excise cassettes. Our results show that IntI1 is more efficient in cassette excision using T-N6-G or T-N6-C attCs while IntI3 recognizes only a limited range of attCs. IntI2*179E and SonIntIA are more tolerant of changes to the identity and spacing of extrahelical bases.ConclusionsThis study provides new insights into the factors that influence the efficiency of cassette excision by integron integrases. It also suggests that IntI2 and SonIntIA have an evolutionary path that is different from IntI1 and IntI3, in their ability to recognize and excise cassettes.

Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double-stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum.

Combining a regeneration-promoting ipt gene and site-specific recombination allows a more efficient apricot transformation and the elimination of marker genes

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In addition, their elimination may allow gene stacking by the same selection strategy. In apricot, selection using the selectable marker gene nptII, that confers resistance to aminoglycoside antibiotics, is relatively effective. An attractive alternative is offered by the MAT system (multi-auto-transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with an MAT vector has been attempted in the apricot cultivar 'Helena'. Regeneration from infected leaves with Agrobacterium harboring a plasmid containing the ipt gene was significantly higher than that from non-transformed controls in a non-selective medium. In addition, transformation efficiencies were much higher than those previously reported using antibiotic selection, probably due to the integration of the regeneration-promoting ipt gene. However, the lack of an ipt expression-induced differential phenotype in apricot made difficult in detecting the marker genes excision and plants had to be evaluated at different times. PCR analysis showed that cassette excision start occurring after 6 months approximately and 1 year in culture was necessary for complete elimination of the cassette in all the transgenic lines. Excision was confirmed by Southern blot analysis. We report here for the first time in a temperate fruit tree that the MAT vector system improves regeneration and transformation efficiency and would allow complete elimination of marker genes from transgenic apricot plants by site-specific recombination.

Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the beta-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the beta-4 strand is less important for the excision activity.

DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development

We here identify a protein (AlfA; actin like filament) that defines a new family of actins that are only distantly related to MreB and ParM. AlfA is required for segregation of Bacillus subtilis plasmid pBET131 (a mini pLS32-derivative) during growth and sporulation. A 3-kb DNA fragment encoding alfA and a downstream gene (alfB) is necessary and sufficient for plasmid stability. AlfA-GFP assembles dynamic cytoskeletal filaments that rapidly turn over (t(1/2)< approximately 45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two-fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation.

Les intégrons en tant que support génétique de résistance aux antibiotiques

Integrons, have been described in the 90's and contain one or more gene cassettes located at a specific site. Integrons are defined by an intI gene encoding an integrase, a recombination site attI and a strong promoter. A gene cassette includes an open reading frame and, at the 3′-end, a recombination site attC. Integration or excision of cassettes occur by a site-specific recombination mechanism catalyzed by the integrase. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in Gram-negative bacteria but integrons in Gram-positive bacteria were described ( Corynebacterium, Enterococcus). Moreover, the finding of super-integrons with gene-cassettes coding for other determinants suggests the likely implication of this multi-component cassette-integron system in bacterial genome evolution before the antibiotic era and in a greater extent than initially believed.

Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be site.

Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron-encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette-associated 59-be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59-be and a non-cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non-cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.

Integrons: an antibiotic resistance gene capture and expression system.

Bacteria can transfer genetic information to provide themselves with protection against most antibiotics. The acquisition of resistance gene arrays involves genetic mobile elements like plasmids and transposons. Another class of genetic structures, termed integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons are defined by an intl gene encoding an integrase, a recombination site attl and a strong promoter. At least six classes of integrons have been determined according to their intl gene. Classes 1, 2 and 3 are the most studied and are largely implicated in the dissemination of antibiotic resistance. A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC. Integration or excision of cassettes occur by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can occur, albeit rarely, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from the common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in gram-negative bacteria but integrons in gram-positive bacteria were described recently. Moreover, the finding of super-integrons with gene-cassettes coding for other determinants (biochemical functions, virulence factors) in Vibrio isolates dating from 1888 suggests the likely implication of this multicomponent cassette-integron system in bacterial genome evolution before the antibiotic era and to a greater extent than initially believed.

The IntI1 integron integrase preferentially binds single-stranded DNA of the attC site.

IntI1 integrase is a member of the prokaryotic DNA integrase superfamily. It is responsible for mobility of antibiotic resistance cassettes found in integrons. IntI1 protein, as well as IntI1-COOH, a truncated form containing its carboxy-terminal domain, has been purified. Electrophoretic mobility shift assays were carried out to study the ability of IntI1 to bind the integrase primary target sites attI and aadA1 attC. When using double-stranded DNA as a substrate, we observed IntI1 binding to attI but not to attC. IntI1-COOH did not bind either attI or attC, indicating that the N-terminal domain of IntI1 was required for binding to double-stranded attI. On the other hand, when we used single-stranded (ss) DNA substrates, IntI1 bound strongly and specifically to ss attC DNA. Binding was strand specific, since only the bottom DNA strand was bound. Protein IntI1-COOH bound ss attC as well as did the complete integrase, indicating that the ability of the protein to bind ss aadA1 attC was contained in the region between amino acids 109 and 337 of IntI1. Binding to ss attI DNA by the integrase, but not by IntI1-COOH, was also observed and was specific for the attI bottom strand, indicating similar capabilities of IntI1 for binding attI DNA in either double-stranded or ss conformation. Footprinting analysis showed that IntI1 protected at least 40 bases of aadA1 attC against DNase I attack. The protected sequence contained two of the four previously proposed IntI1 DNA binding sites, including the crossover site. Preferential ssDNA binding can be a significant activity of IntI1 integrase, which suggests the utilization of extruded cruciforms in the reaction mechanisms leading to cassette excision and integration.

Cre/loxP-mediated excision of a neomycin resistance expression unit from an integrated retroviral vector increases long terminal repeat-driven transcription in human hematopoietic cells.

Recombinant retroviruses are currently the most attractive vehicles for gene transfer into hematopoietic cells. Retroviral vectors often contain an easily selectable marker gene in addition to the gene of interest. However, the presence and selection for expression of the selectable gene often result in a significant reduction of the expression of the gene of interest in the transduced cells. In order to circumvent this problem, we have developed a Cre/loxP recombination system for specific excision of the selectable expression unit from integrated retroviruses. A retroviral vector, containing both a neomycin resistance expression unit flanked by loxP sites and granulocyte-macrophage colony-stimulating factor cDNA, was used to transduce the human hematopoietic K-562 cell line. Four transduced cell clones were then superinfected with a retrovirus containing a Cre recombinase expression unit. Molecular analyses of 30 doubly transduced subclones showed a strict correlation between cre expression and loxP-flanked selectable cassette excision, thus implying that Cre recombinase activity is very efficient in a retroviral context. Moreover, the excision of the selectable cassette results in a significant increase of granulocyte-macrophage colony-stimulating factor transcription driven by the retroviral promoter.

Gene cassettes from the insert region of integrons are excised as covalently closed circles.

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.