DNA Cassette Research Articles

547. AAV Preparations Contain Contamination from DNA Sequences in Production Plasmids Directly Outside of the ITRs

Despite having the best safety profile of any current clinical viral vector, it is known that AAV preps contain contaminating sequences that are packaged alongside the expression cassette at a low rate. These sequences can originate from production plasmid DNA, or chromosomal DNA from producer cell lines. It has been reported that sequences from the expression cassette producer plasmid are more likely to be packaged into AAV than DNA from other producer plasmids, or chromosomal DNA. We hypothesised that in the expression cassette plasmid, backbone sequences directly flanking the ITRs might be packaged into AAV at a higher rate than sequences further away from either ITR. We first confirmed the presence of these sequences via PCR amplification of non-expression cassette DNA flanking the ITR sequence up to 1.5kb in length in an AAV prep. Sequential qPCR assays showed that plasmid sequences at a range of distances up to 2kb from the ITR make up between 1% and 9% of AAV particles. Most significantly, there was an observable decreasing trend in contaminant titer as distance from the ITR increased. Contaminant sequences closer to the ITRs (within 1kb) are detected at a 100 fold greater rate than distal plasmid DNA (9kb from ITRs on the same plasmid). The disparity in the levels of ITR adjacent DNA sequences, compared to sequences 9kb from either ITR, suggest that the origin of this DNA is from within AAV particles rather than residual plasmid DNA remaining after purification procedures. ITR adjacent contamination is present at both a TRS mutated ITR (required for self-complementary vectors) and non TRS mutated ITRs. Contaminating plasmid sequences were present when the transgene was half of the packaging capacity (2.3kb FIX prep) and at the full capacity (5kb FVIII prep) at comparable levels, suggesting that increasing the transgene size with stuffer DNA to create a full genome will not solve this issue. Previous studies have concluded that increasing the size of the backbone with stuffer DNA reduces the level of plasmid backbone contamination, as the two ITRs are then not in range of each other to facilitate reverse packaging. However, the total plasmid size in our studies was >20kb. Therefore, the ITRs should not be in range for this to occur. We hypothesise that these ITR adjacent sequences are either a product of read-through from the expression cassette into flanking sequences, due to inefficient cleavage at the ITR breakpoint, or from reverse priming mediated by only 1 functional ITR. In the current expression cassette plasmids examined, the Kanr gene and bacterial f1origin of replication are within the range of the flanking sequences that could be packaged. With current, unsolved clinical challenges for AAV, including transaminitis post high dose infection, it is clear that clinical AAV vectors should be designed to contain as little contamination as possible. We conclude that newly designed AAV production plasmids should contain significant lengths of stuffer DNA flanking each ITR (at least 2kb) to ensure that bacterial sequences are not packaged into AAV preps. Further research into vector design is required to eliminate this source of non-functional DNA from AAV produced for the clinic.View Large Image | Download PowerPoint Slide

Modelling the in-utero initation of ETV6-RUNX1 in childhood acute lymphoblastic leukaemia using human pluripotent stem cells

BackgroundWe hypothesise that the clinical differences between adult and childhood acute lymphoblastic leukaemia (cALL) arise partly through their origin in developmentally distinct target cells. Since cALL frequently initiates in utero we aimed to characterise the earliest stages of lymphoid development in human fetal liver. In parallel we used human pluripotent stem cells (hPSC) to recapitulate fetal liver lymphopoiesis and model the impact of the fusion oncogene ETV6-RUNX1, the commonest genetic aberration in cALL. MethodsH1 human embyronic stem cells and MIFF3 human induced hPSCs (provided by University of Sheffield Centre for Stem Cell Biology) were maintained in vitro with mTeSR1/Matrigel. Vectors were produced by recombineering an ETV6 bacterial artificial chromosome with a custom DNA cassette (GeneArt, ThermoFisher, Waltham MA, USA). Vectors were transfected (Nucleofector II, Lonza, Basle, Switzerland) and G418-selected clones were screened by Southern blot. hPSCs were differentiated by sequential OP9/MS5 coculture. Populations sorted by fluorescence-activated cell sorting were analysed by single cell real-time PCR (Biomark 48.48, Fluidigm, San Fransisco, CA, USA) and 200 cell RNA sequencing. Human fetal livers were donated under informed consent with approval of Lund University Ethical Review Board and the Swedish National Board of Health and Welfare. FindingsB lymphopoiesis in the fetal liver was distinct from that in adult bone marrow or neonatal cord blood: the earliest fetal liver B cells did not fully express mature lymphoid effectors and the earliest identified lymphoid-capable progenitors coexpressed both lymphoid and myeloid gene expression programmes. Global and single cell gene expression profiling of B lymphopoietic progenitors generated by in vitro differentiation of hPSCs showed that they share the same transcriptional programme. hPSCs that were CRISPR-engineered to express ETV6-RUNX1 were partly arrested in B cell differentiation at the level of the lymphomyeloid progenitor. B cells that passed this block shared the global gene expression signature of the fetal lymphomyeloid progenitor, and single cell real-time PCR showed that they aberrantly coexpressed B and myeloid lineage genes. InterpretationOur results identify a B lymphoid progenitor in human fetal liver characterised by coexpression of lymphoid and myeloid gene expression programmes and suggest that this progenitor's B myeloid signature provides a permissive transcriptional context for ETV6-RUNX1 to effect a partial differentiation arrest. The developmental specificity of the gene expression signature of this cell could offer unique therapeutic targets, and the hSPC model might provide a novel drug-screening platform. FundingWellcome Trust Research Training Fellowship and NIHR Academic Clinical Fellowship (SR); Swedish Childhood Cancer Foundation (CB); Bloodwise, Cancer Research UK, Children with Cancer, and Great Ormond Street Hospital Children's Charity (TE).

Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment

We have developed a method for marine diatom transformation by microparticle bombardment using polymerase chain reaction (PCR)-amplified DNA fragments. We constructed a circular vector (approximately 5000bp) containing an fcpA promoter from Phaeodactylum tricornutum, antibiotic-resistance genes and terminator from Cylindrotheca fusiformis (a “gene cassette”). Then the various lengths of linear vectors (+0–+1000 linear vectors) were then PCR-amplified from the circular plasmid. The transformants of P. tricornutum transfected with the linear vectors were obtained in the triplicate experiments. Transformation efficiencies using PCR-amplified short linear vectors containing the gene cassette and additional DNA regions of 0, 50, and 500bp at both ends of the gene cassette (+0–+500 linear vectors) did not significantly differ from one another or from the efficiency of the +1000 linear vector. Transformation efficiencies using the linear vectors were lower than that using the circular vector, but were not significantly different. The ratios of the number of transformants containing the whole region of the gene cassette to those of transformants transfected using linear vectors of various lengths were determined. An extension (≧50bp) of DNA fragments was effective for introducing the whole region of the gene cassette into the genomic DNA. In using various amounts of the +50 linear vector (37.5–300fmol/shot), we observed that transformation efficiencies using 37.5fmol (52.2ng)/shot of the linear vector were not significantly different from those obtained using 300fmol of the linear vector. The 300fmol quantity was set considering the quantity of the circular plasmid (1μg=approx. 300fmol) and the 37.5fmol quantity was set for quick and easy preparation of approximately 500ng of the linear short vector needed for triplicate transformation experiments in one PCR tube containing 50μl of PCR cocktail. Integrating the gene cassette of the short linear vectors as well as that of the full length of the linear vector (+1000 linear vector) into the chromosomal DNA was determined using Southern blot analysis. The short linear vectors tended to result in smaller numbers of insertions than those of the supercoiled plasmid. This simple and time-saving transformation method using microparticle bombardment with PCR-amplified DNA fragments permitted both functional analysis of diatom-specific genes and development of diatom strains useful for further biotechnological applications.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Use of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia coli

Various methods have been developed for gene disruption in bacteria; however, extra in vitro manipulation steps or the residual presence of a scar in the host chromosome limits the use of such methods. By utilizing the unique properties of ISHp608, we have developed a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The λ Red mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. We demonstrated lacZ gene point mutation repair, two precise disruptions of the lacZ gene and constructed a library of lacZ variants having variable β-galactosidase activity by changing its ribosome binding site sequences using the ISHp608 system. This technique can be used in E. coli genome modification and could be extended for use in other bacteria.

English

Although microalgae have valuable resources with great necessity in many biotechnological applications, no tools have been developed yet for a stable genetic transformation without antibiotic marker genes in these organisms. Chlorella is one of the most useful genus for biotechnological applications. The transfer of foreign DNA (vector or linear DNA cassette) into Chlorella by electroporation has very low stability and it is hard to screen the transformants without antibiotic marker genes. However, the marker genes have some disadvantages to host cells. To avoid the negative effects caused by the marker genes, we tried to develop a stable marker-free nuclear transformation system in Chlorella. For this, linear gene expression cassettes (LGEC) were constructed with functional domains, which are responsible for transformation, of SV40 large T antigen. The LGECs were transferred into Chlorella via electroporation and durability of the LGECs were confirmed in transgenic Chlorella. Transcription levels of the LGECs were also determined at different cell cycle sates. The LGECs integrated into the chromosomal DNA of Chlorella were stably replicated and were expressed successfully at G0-, G1-, and G2-phases. This study presents a stable marker-free nuclear transformation system with potential for biotechnological applications. Key words: Chlorella vulgaris, marker-free nuclear transformation, SV40 large T antigen, microalga.

Metabolic engineering of E. coli for efficient production of glycolic acid from glucose

Glycolic acid is the smallest member of the α-hydroxy acid family. In order to produce glycolate from glucose via the glyoxylate shunt stably, one malate synthase gene aceB in Escherichia coli BW25113 was deleted by homologous recombination; another malate synthase gene glcB was then replaced by a DNA cassette WAK harboring isocitrate lyase gene (aceA), glyoxylate reductase gene (ycdW) and isocitrate dehydrogenase kinase/phosphatase gene (aceK). The above three genes were over-expressed in the chromosome of E. coli EYX-1WAK. This strain was then transferred 20 times on M9 medium to have a mutant strain: EYX-2 with a significantly improved growth rate. The glycolate yields of EYX-2 in the shaken flasks and the 5-L bioreactor using batch fermentation strategy under 2vvm aeration and 800rpm stirring speed were 0.33g/g-glucose and 0.48g/g-glucose, respectively. The fed-batch fermentation of EYX-2 on 120g/L glucose had the highest titer of 56.44g/L with 0.52g/g-glucose yield in 120h, and this is the highest reported glycolate yield ever.

I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6829-1) contains supplementary material, which is available to authorized users.

Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice

Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone™ linear closed DNA [(dbDNA™)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4+ and CD8+ T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA™ and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches.

Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

Differential requirements of singleplex and multiplex recombineering of large DNA constructs.

Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.

686. Gene Correction of IL2RG in Human Hematopoietic Stem and Progenitor Cells

Mutations in the IL2RG gene may result in X-linked severe combined immunodeficiency (X-SCID), an immunodeficiency disorder in which the body produces very few T cells and natural killer cells, resulting in premature death due to opportunistic infections. At present, allogeneic hematopoietic stem cell transplant (HSCT) is the standard of care and results in full immune reconstitution if engraftment is successful. However, the search for a compatible donor is not always successful and transplantation from a non MHC-identical donor carries the risk of graft-versus-host disease. An autologous HSCT containing a replaced or corrected IL2RG gene would avoid these risks.Previous autologous gene therapy for X-SCID successfully treated the disease but relied on early generation gene replacement retroviral vectors and resulted in insertional oncogenesis and leukemia development in some treated subjects. Here we report a gene correction approach to X-SCID gene therapy involving targeted integration (TI) of a corrective IL2RG donor DNA cassette (cDNA from exons 2-8) into intron 1 of the IL2RG gene. This strategy has the advantage of retaining the endogenous IL2RG promoter and enhancer and allows treatment of patients with almost all X-SCID causing IL2RG mutations (excluding only those affecting the gene upstream of intron 1). Using engineered zinc-finger nucleases (ZFNs) delivered as mRNA and IL2RG donor DNA delivered as adeno-associated virus (AAV) to CD34+ human hematopoietic stem and progenitor cells (HSPCs) cultured ex vivo, we have achieved TI of the corrective IL2RG donor in up to 25% of alleles. These cells differentiate normally in vitro and retain consistent levels of TI in both erythroid and myeloid lineages. IL2RG mRNA expression was successfully restored in an IL2RG knockout model human cell line, validating this strategy. Coupled with the strong, intrinsic in vivo selection for cells with IL2RG activity, our data are a promising advance towards an autologous HSPC-based gene therapy for the treatment of X-SCID.

Puromycin- and methotrexate-resistance cassettes and optimized Cre-recombinase expression plasmids for use in yeast.

Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin-resistance can be achieved in yeast through expression of a bacterial puromycin-resistance gene optimized to the yeast codon bias, which in turn serves as an easy-to-use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon-optimized mutant human dihydrofolate reductase (DHFR), which confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug-resistant marker cassettes are flanked by loxP sites, allowing for their excision from the genome following expression of Cre-recombinase. Finally, we have created a series of plasmids for low-level constitutive expression of Cre-recombinase in yeast that allows for efficient excision of loxP-flanked markers.

Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated asimilar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.

A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system.

The broad host range of pseudorabies virus (PRV) and large capacity for foreign DNA make it a promising vector for the development of vaccines and agents of gene therapy. We show that up to 100 % viral gene disrupting efficiency was achieved by simple co-transfection of the purified PRV genomes with the clustered regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) into cells. Furthermore, CRISPR/Cas9-mediated knock-in of >4-kb-long DNA cassettes into the PRV genome at a positive rate of 50 % by a homology-independent DNA repair mechanism without constructing homology arms. This approach requires only a simple plasmid construction and is applicable to knock-in of other foreign genes. Our studies offered simple and efficient methods to manipulate PRV.

Cre Recombinase.

The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases. The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts, has been an important factor in both cases. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.

An improved inverse PCR protocol for the generation of T-vectors

T-vectors have been widely used in molecular cloning [1–3]. Compared with common vectors, molecular cloning with T-vectors escapes the restriction endonuclease digestion process, decreases the time of DNA purification and thus is time-saving and cost-effective. There are two ways to produce T-vectors: (i) cutting the target vector with EcoRV and then adding a thymine residue to the 3′-blunt end with TaqDNA polymerase [4]; and (ii) inserting a DNA cassette containing XcmI or AhdI recognition sites at its both ends and then splitting the recombinant vector with them later [5–7]. However, only a small amount of the T-vector can be produced by these methods. What’s more, the residual undigested primary vector may cause false-positive clones. Therefore, T-vectors used in labs are usually bought from biotechnology companies providing with small amounts, thus limiting their application in conventional molecular cloning experiments. Here, we designed an improved inverse PCR protocol for the generation of T-vectors (Fig. 1). Briefly, an inverse PCR was performed with the target vector as a template with a pair of phosphorylated primers, then the PCR products were subject toDpnI treatment, agarose gel purification and further, the terminal deoxynucleotidyl transferase was applied to add a T to the 3′-blunt end of the PCR products which will ligate to the PCR products of the gene of interest with 3′-A overhangs. Afterwards, the ligation mixture was transformed into Escherichia coliDH5α competent cells, and a routine PCR was performed to calculate the positive rate of the transformed clones. The positive clones were further confirmed by DNA sequencing. Finally, we got at least 90% positive clones with our improved protocol. The main materials used in this study includes: the pcDNA3.0 vector, pcDNA3.0-FLAG vector containing human histone H4 cDNA, pcDNA3FLAG-CRBN vector all from our laboratory, PfuUltra II Fusion HS DNA polymerase from Stratagene (Cedar Creek, USA), the E. coli DH5α competent cells, DpnI, premix Taq, and T4 polynucleotide kinase (T4 PNK) fromTaKaRa Biotechnology (Dalian, China), ddTTP fromSigma-Aldrich (St Louis, USA),TaqDNApolymerasewith ThermoPol buffer, terminal deoxynucleotidyl transferase (TdT) from New England Biolabs (NEB; Massachusetts, USA), agarose gel DNA purification kit and PCR purification kit are all from Tiangen Biotech (Beijing, China). The sequence of the primer set for inverse PCR is as follows: forward primer 5′-TTCTATAGTGTCACCTAAATGCTAGA GC-3′, and reverse primer 5′-CCTATAGTGAGTCGTATTAATTT CGATAA-3′. The sequences of the primer pair for FLAGand human histone H4 fusion cDNA are as follows: forward primer 5′-CGGAATT CGCCACCATGGACTACAAGGACGACGA-3′; and reverse primer 5′-C GGGATCCACCGCCGAAACCATAAAGGG-3′; and the sequence of the primer pair for human CRBN cDNA is as follows: forward primer 5′-CCCAAGCTTATGGCCGGCGAAGGAGATC-3′; and reverse primer 5′-CGGGATCCTTACAAGCAAAGTATTACTTTGTC-3′. The T7 and SP6 primer pair was employed in routine PCR to evaluate the positive rate of the transformed clones. The DNA sequence of T7 primer is: 5′-TAATACGACTCACTATAGG-3′. The DNA sequence of SP6 primer is: 5′-ATTTAGGTGACACTATAGAA-3′. DNA sequencing was accomplished with the following primers: forward primer 5′-TTT CCAAAATGTCGTAACAACT-3′, and reverse primer 5′-AGACAATG CGATGCAATTTC-3′. All the above-mentioned primers were synthesized in Sangon Biotech Co., Ltd (Shanghai, China). To determined its usability, the T-vector generated from pcDNA3.0 vector was ligated to the coding sequence of FLAG tag and human histone H4 fusion protein (387 bp) as well as to the cDNA of human CRBN (1329 bp). The positive rate of the transformed clones was evaluated by routine PCR and the rightness of the insertion fragments was confirmed by DNA sequencing. Before the inverse PCR was performed, both the forward and reverse primers were phosphorylated in 20 μl reaction solution containing 5 μl primer (10 μM), 2 μl dNTP (10 mM), 2 μl T4 PNK buffer, 1 μl T4 PNK, and 10 μl distilled water. For inverse PCR, the following reagents were added into a 200 μl PCR tube: 10 ng pcDNA3.0 template plasmid, 400 μM dNTP, 0.8 μM forward and reverse primers respectively, 0.5 μl PfuUltra II Fusion HS DNA polymerase and distilled water was supplemented to 50 μl. Each PCR thermal cycle includes Acta Biochim Biophys Sin, 2015, 47(2), 142–144 doi: 10.1093/abbs/gmu118 Advance Access Publication Date: 23 December 2014 Lab Note

Construction of Adoxophyes honmai nucleopolyhedrovirus bacmid DNA by using a gene-repair BAC cassette

Recombinant baculoviruses have traditionally been generated by homologous recombination, in insect cells, of the virus DNA and a transfer vector. This method is, however, restricted to the small number of baculoviruses for which permissive cell lines are available. An alternative method, which avoids this limitation, is the bacmid system, in which a bacterial DNA cassette (BAC cassette) comprising a bacterial replication origin, an antibiotic resistance marker gene, and a transposon element, is inserted into a unique restriction endonuclease site of the virus genome and the recombinant DNA is produced in Escherichia coli K-12 strain. However, if the unique cleavage site is located within an open reading frame (ORF), the ORF will be disrupted by BAC cassette insertion. In this study, we propose a gene-repair BAC method which simultaneously restores the disrupted ORF and inserts a BAC cassette into the full genome of Adoxophyes honmai nucleopolyhedrovirus.

Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.