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The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes.

The bicistronic design (BCD) is characterized by a short fore-cistron sequence and a second Shine-Dalgarno (SD2) sequence upstream of the target gene. The outstanding performance of this expression cassette in promoting recombinant protein production has attracted attention. Recently, the application of the BCD has been further extended to gene expression control, protein translation monitoring, and membrane protein production. In this review, we summarize the characteristics, molecular mechanisms, applications, and structural optimization of the BCD expression cassette. We also specifically discuss the challenges that the BCD system still faces. This is the first review of the BCD expression strategy, and it is believed that an in-depth understanding of the BCD will help researchers to better utilize and develop it. KEY POINTS: • Summary of the characteristics and molecular mechanisms of the BCD system. • Review of the actual applications of the BCD expression cassette. • Summary of the structural optimization of the BCD system.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

Aims The aim of this article is to study the functional features of Penicillium oxalicum transcriptional activator XlnR. Methods and results The yeast reporter system was used to identify transcriptional activation domain of XlnR in P. oxalicum. The expression cassette was introduced into the xlnR locus of P. oxalicum by homologous recombination. In this study, several putative structural domains in P. oxalicum XlnR were predicted by bioinformatics analysis, and the transcriptional activation domain (351-694 region) was identified in XlnR relying on reporter gene system in yeast. In addition, the amino acid at XlnR 871 site (alanine) located in the regulatory region could influence the regulatory activity of XlnR directly. When the alanine at XlnR 871 site was replaced by stronger hydrophobic amino acid (e.g. valine or isoleucine), the regulatory activity will be greatly improved, especially for the regulation of hemicellulase genes expression. When alanine at XlnR 871 site was mutated to a hydrophilic amino acid (e.g. aspartic acid or arginine), the regulatory activity of XlnR will be reduced. Conclusions The 351-694 region of P. oxalicum XlnR was identified as transcriptional activation domain, and the regulatory activity of XlnR was greatly influenced by hydrophobicity of amino acid at 871 site of XlnR in P. oxalicum. Significance and impact of the study The results will provide an effective target site to regulate the activity of XlnR and improve cellulase production of P. oxalicum.

Napier grass (Cenchrus purpureus Schum.) is one of the highest yielding tropical C4 forage grasses and a promising candidate feedstock for the production of biofuel. Genetic engineering and genome editing have tremendous potential for crop improvement and require the establishment of a transformation protocol. In this study, transgenic Napier grass was generated by introducing a minimal cassette (MC) encoding the antibiotic resistance gene nptII with biolistic gene transfer into embryogenic callus. Paromomycin was superior to geneticin as a selective agent in the selection of transgenic calli. Two high-biomass producing Napier grass accessions were successfully transformed. Southern blot analysis revealed simple transgene integration patterns with 33% of the regenerated transgenic lines having a single insert of the minimal transgene expression cassette. NPTII-ELISA confirmed transgene expression in all transgenic lines. The results of this study will enable the acceleration of Napier grass improvement by genetic transformation or genome editing.

Hepatic gene transfer with adeno-associated viral (AAV) vectors shows much promise for the treatment of the X-linked bleeding disorder hemophilia B in multiple clinical trials. In an effort to further innovate this approach and to introduce alternative vector designs with potentially superior features into clinical development, we recently built a vector platform based on AAV serotype 3 because of its superior tropism for human hepatocytes. A vector genome with serotype-matched inverted terminal repeats expressing hyperactive human coagulation factor IX (FIX)-Padua was designed for clinical use that is optimized for translation using hepatocyte-specific codon-usage bias and is depleted of immune stimulatory CpG motifs. Here, this vector genome was packaged into AAV3 (T492V + S663V) capsid for hepatic gene transfer in non-human primates. FIX activity within or near the normal range was obtained at a low vector dose of 5 × 1011 vector genomes/kg. Pre-existing neutralizing antibodies, however, completely or partially blocked hepatic gene transfer at that dose. No CD8+ T cell response against capsid was observed. Antibodies against the human FIX transgene product formed at a 10-fold higher vector dose, albeit hepatic gene transfer was remarkably consistent, and sustained FIX activity in the normal range was nonetheless achieved in two of three animals for the 3-month duration of the study. These results support the use of this vector at low vector doses for gene therapy of hemophilia B in humans., Graphical abstract, Hepatic AAV gene transfer shows much promise for the treatment of hemophilia. Here, a vector with AAV3 serotype-matched inverted terminal repeats expressing coagulation factor IX-Padua was designed using hepatocyte-specific codon bias and depletion of CpG motifs. FIX activity near the normal range was obtained at low vector dose in non-human primates.

Background Bispecific antibodies represent an important class of monoclonal antibodies (mAbs), with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti-CD3 × CD19 bispecific antibody (bsAb) is a bispecific T-cell engager currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods woodchuck hepatitis virus post-transcriptional regulation (WPRE) sequence was incorporated at the 3' end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & conclusion Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in human embryonic kidney-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody.

Biodistribution of self-complementary adeno-associated virus-9 (scAAV9)–chicken β-actin promoter–green fluorescent protein (GFP) was assessed in juvenile cynomolgus macaques infused intrathecally via lumbar puncture or the intracisterna magna (1.0×1013 or 3.0×1013 vg/animal), with necropsy 28 days later. Our results characterized central nervous system biodistribution compared with systemic organs/tissues by droplet digital polymerase chain reaction for DNA and in situ hybridization. Green fluorescent protein expression was characterized by Meso Scale Discovery electrochemiluminescence immunosorbent assay and immunohistochemistry (IHC). Biodistribution was widespread but variable, with vector DNA and GFP expression greatest in the spinal cord, dorsal root ganglia (DRG), and certain systemic tissues (e.g., liver), with low concentrations in many brain regions despite direct cerebrospinal fluid administration. Transduction and expression were observed primarily in perivascular astrocytes in the brain, with a paucity in neurons. Greater GFP expression was observed in hepatocytes, striated myocytes, cardiomyocytes, spinal cord lower motor neurons, and DRG sensory neurons by IHC. These results should be considered when evaluating scAAV9-based intrathecal delivery with the current expression cassette as a modality for neurologic diseases that require widespread brain neuronal expression. This capsid/expression cassette combination may be better suited for diseases that express a secreted protein and/or do not require widespread brain neuronal transduction.

Robust and versatile promoters for Lactobacillus plantarum found in wine are necessary gene expression tools for genetic research involving wine stress. We optimized the electrotransformation parameters for L. plantarum XJ25 isolated from wine and engineered five promoters based on the promoter P23; these promoters showed significantly different transcriptional activities under nonstress conditions. The activities of these promoters in vivo and the resulting growth burden to the host strain under different wine stresses were also evaluated. A range of colors (from white to dark pink) of the developing colonies with the plasmid pNZ8148 carrying an X-mCherry expression cassette, namely, P23-mCherry, trcP23-mCherry, POL1-mCherry, POL2-mCherry, POL3-mCherry, or POL4-mCherry, were analyzed. The applicability of the optimized electrotransformation parameters and synthetic promoters with different activities were also verified in several L. plantarum strains. Therefore, the optimized electrotransformation and these characterized promoters were determined to be suitable for applications in wine research in the future.

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype’s characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

Streptomyces transglutaminase (TGase) is widely used to improve food texture properties. In this study, random mutagenesis and site-directed genetic modification were used to improve the production of TGase in Streptomyces mobaraensis. First, S. mobaraensis DSM40587 (smWT) was subjected to atmospheric and room-temperature plasma mutagenesis, and then a mutant (smY2019) with a 5.5-fold increase in TGase yield was screened from approximately 3000 × 25 (round) mutants. Compared to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two site mutations within the -10 region of the TGase promoter. The recombinant expression analysis in the TGase-deficient S. mobaraensis suggests that the mutated TGase promoter is more robust than the wild-type one. Finally, we integrated two additional TGase expression cassettes into the smY2019 genome, yielding the recombinant strain smY2019-3C with a 103% increase in TGase production compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield could be a suitable candidate for the industrial production of TGase.

The codon-optimized zebrafish β-defensin 3 mature peptide gene mzfDB3 and channel catfish c-type lysozyme gene cflyC were used to design the fusion gene cflyC-mzfDB3. In the fusion protein cflyC-mzfDB3, 4×Gly flexible amino acid linker with an enterokinase cleavage site DDDDK was designed to link the mzfDB3 to the C-terminus of cflyC, which potentially contributes to digestion of the recombinant cflyC-mzfDB3 by endogenous enterokinases. The fusion protein was expressed in Pichia pastoris X-33 using expression vectors harboring 1-, 2-, or 4-copies, respectively, of the expression cassette. The cflyC-mzfDB3 gene copy numbers of P. pastoris transformants were quantified using real-time quantitative PCR. Their expression yields showed that the expression level in the 4-copy cflyC-mzfDB3 transformant was 2.67-fold higher than that in the 1-copy cflyC-mzfDB3 transformant, demonstrating that the increase in the cflyC-mzfDB3 gene copy number resulted in increased protein expression. The culture medium supernatant containing recombinant cflyC-mzfDB3 exhibited antibacterial activity against Gram-positive Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa, indicating that there may be a synergistic effect of mzfDB3 and cflyC on the peptide’s antibacterial activity. In addition, cell-free P. pastoris medium may be a potential candidate for further development as a natural hybrid antimicrobial solution.

Red rot caused by Colletotrichum falcatum, is one of the economically important disease of sugarcane and breeding for resistant varieties is considered to be the major solution to manage the disease. However, breakdown of red rot resistance become usual phenomenon due to development of newer races by culture adaptation on newly released varieties. Hence it is needed to characterize the genes responsible for pathogen virulence in order to take care of host resistance or to manage the disease by other methods. The transcript studies gave foundation to characterize the huge number of pathogenicity determinants and their role in pathogenesis. Here we studied role of two important genes viz., Glucose Transporter (GT) and Sucrose Non-Fermenting1 (SNF1) during pathogenesis of C. falcatum, which said to be involved in carbon source metabolism. Sugar metabolism has a vital role in disease progression of C. falcatum by regulating their cell growth, metabolism and development of the pathogen during various stages of infection. The present study was aimed to find out the role of GT and SNF1 genes in response to pathogenicity by RNA silencing (RNAi) approach. Knock-down of the target pathogenicity gene homologs in standard C. falcatum isolate Cf671 was carried out by amplifying sense and antisense fragments of targets individually using pSilent-1 vector. The expression cassette was cloned into the binary vector pCAMBIA1300 followed by fungal transformation through Agarobacterium mediated transformation. Resulted mutants of both the genes showed less virulence compared to wild type isolate. Simultaneously, both the mutants did not produce spores. Moreover, the molecular confirmation of the mutants displayed the expression of hygromycin gene with reduced expression of the target gene during host-pathogen interaction. Knockdown of the pathogenicity related genes (GT and SNF1) by RNAi approach corroborate the possible role of the genes in causing the disease.

Gene editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems have been developed to create targeted DNA mutagenesis in many crop plants. This report describes application of the TALEN system to generate bialaphos resistance (bar)-knockout null segregants in herbicide-tolerant rice (Ba15) and microarray analysis on transcriptome changes of mutated lines, to identify unexpected effects resulting from off-targets. We generated 41 T0 plants and identified TALEN-mediated bar sequence mutations in 14 of them. Non-target site single nucleotide polymorphisms (SNPs) and small insertion/deletions (InDels) accounted for a large proportion of the mutations. Segregations of phosphinothricin acetyltransferase (PAT) protein expression levels were observed in T1 generations of two lines, R6 and R9. In addition, most T1 offspring harbored the TALE-R expression cassette and acquired some de novo mutations that were not inherited from their T0 parents. Three bar-knockout T1 lines were tested for PAT protein expression in progeny seedlings, and their T2 plants possessed inactive bar. We selected three bar-knockout T2 plants that were TALE-DNA-free for microarray analysis, aiming to understand the transcriptome differences between mutated null segregants and their recipient line. Only 31 differentially expressed genes (DEGs) were identified in the bar-knockout rice lines, possibly resulting from somaclonal variations from the in vitro cell culture process. Taken together, TALEN-mediated bar mutations have little effect on the whole transcriptome profile of rice. We believe our results will be helpful to study unexpected consequences in gene-edited crops.

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under β-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.

A transgenic acute promyelocytic leukemia (APL) murine model established by Michael Bishop by cloning a human PML-RARα cDNA into the hMRP8 expression cassette has been widely used in the all-trans ...

Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.

As a biorefinery platform host, Escherichia coli has been used extensively to produce metabolites of commercial interest. Integration of foreign DNA onto the bacterial genome allows for stable expression overcoming the need for plasmid expression and its associated instability. Despite the development of numerous tools and genome editing technologies, the question of where to incorporate a synthetic pathway remains unanswered. To address this issue, we studied the genomic expression in E. coli and linked it not only to 26 rationally selected genomic locations, but also to the gene direction in relation to the DNA replication fork, to the carbon and nitrogen source, to DNA folding and supercoiling, and to metabolic burden. To enable these experiments, we have designed a fluorescent expression cassette to eliminate specific local effects on gene expression. Overall it can be concluded that although the expression range obtained by changing the genomic location of a pathway is small compared to the range typically seen in promoter-RBS libraries, the effect of culture medium, environmental stress and metabolic burden can be substantial. The characterization of multiple effects on genomic expression, and the associated libraries of well-characterized strains, will only stimulate and improve the creation of stable production hosts fit for industrial settings.

Modern studies of detailed processes in plant development would not be possible without using a wide range of fluorescent proteins (R. Day and M. Davidson, 2009; D. Chudakov et al., 2010). However, application of fluorescent proteins is restricted due to problems with their visualization in plant tissues. Plants are difficult objects for microscopic studies: even the most advanced methods have significant limitations regarding the depth of light penetration due to the scattering and absorption of light by cell walls. Therefore, to study the distribution of reporter fluorescent proteins in large organs typical for most plants, it is necessary to fix the plant material and prepare thick histological sections with a vibrating-blade microtome. Chemicals traditionally used for fixing, dehydrating and embedding of plant tissues samples lead to changes in the structure of fluorescent proteins and, as a result, often to the loss of their fluorescence. Therefore, it is important to optimize the protocols for fixing plant tissues, preparing sections and studying the distribution of fluorescent proteins by laser scanning confocal microscopy. In this work, we have proposed for the first time an integrated approach to fixation of tissues of transgenic plants and preparation of sections in the course of the study of patterns of cellular response to auxin and expression of transcription factors using laser scanning confocal microscopy. Our aim was to sum up modern approaches to the application of an effective universal technique for visualization of tissue and cellular patterns of the fluorescent reporter proteins distribution on sections of large non-model plants. The first step for using fluorescent proteins in plants is the generation of genetic constructs that carry the promoter of the gene of interest fused to a reporter gene encoding a fluorescent protein. In this case, a transformation protocol has to be available for the selected plant species. We have described the use of the Gateway® cloning technology for the construction of vectors for plant transformation that meet modern experimental requirements. In order to study auxin localization in vivo we developed a series of vectors with genes encoding various fluorescent proteins (eGFP, tdTomato, mRuby3) under the control of the auxin-sensitive DR5 promoter (E. Ilina et al., 2012). We now demonstrate the advantage of nuclear-targeted fluorescent proteins (mNeonGreen-H2B, tdTomato-H2B, mRuby3-H2B), as well as the possibility of their application for additional visualization of cell nuclei in combination with highly specific cell wall staining using SCRI Renaissance2200. An effective method for constructing vectors to study cell-specific expression patterns of developmental regulators is presented using the transcription factor genes GATA24 (A. Kiryushkin et al., 2019) and LBD16 in some Cucurbitaceae species as examples. We also applied an expression cassette, pAtUBQ10::DsRED1 (E. Limpens et al., 2004), carrying a gene encoding red fluorescent protein under the control of a constitutive promoter, to demonstrate the advantages of the use of fluorescent proteins in screening for transgenic vs. wild type roots. A new method of fixation and clearing of plant tissues containing reporter fluorescent proteins and preparation of sections is presented for the example of transgenic roots of Cucurbitaceae. The advantage of using melted agarose compared to embedding media for orienting plant parts during the preparation of samples was demonstrated. The increase of the photostability of fluorescent proteins in sections due to the use of clearing reagent ClearSee (D. Kurihara et al., 2015) as a mounting medium is demonstrated. Altogether, we present a combination of the advantages that are provided by application of a complex of several modern methodical approaches of sample preparation and laser scanning confocal microscopy that offers significant advantages for studying of the developmental biology of large non-model plants.

This review will summarize ocular gene therapy clinical trials and associated technology that have been initiated within the last 5 years. Initial programs utilized subretinal administration of an AAV2 serotype coupled with a ubiquitous promoter and a transgene targeting both monogenic inherited retinal diseases and acquired chronic ocular diseases polygenic in origin such as age-related macular degeneration and diabetic retinopathy. Cellular specific viral vectors with optimization of the expression cassette to include cellular-specific promoters and cassette regulatory elements to improve yields and durability with less invasive administration routes are now being clinically evaluated. These developments open the door for potential treatment of rare monogenic diseases and more common polygenic heterogeneous ocular disorders, where in situ expression of therapeutic proteins may reduce the need for repeated intravitreal injections. While initial reports are promising, only time will confirm whether sustained durable “curative” expression is a reality.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been extensively used for gene editing of many plant species, including model plants such as Arabidopsis and rice, and major crops such as maize and wheat, as well as woody plants like poplar, bamboo, apple and citrus, etc. This technique has not only significantly promoted basic research, but has also facilitated the agronomic traits improvement of economically important crops or trees. Typically, the CRISPR/Cas system required its integration and expression of exogenous DNA in the genome of host plants, which lead to the cleavage of site-specific DNA double-strand breaks, and introduced the genome modifications during the DNA repair process. However, the integration of CRISPR/Cas elements brings several concerns for commercialization and basic research. First, the inserted CRISPR/Cas cassette is constitutively expressed in plant cells, and may increase the chance of off-target changes, as well as make it difficult to evaluate the stability and penetrance of the initially observed phenotypes. Second, plants with a foreign gene integrated into its genome were regarded as classic genetically modified organisms, which may potentially lead to the escape of the gene into the environment, leading to legislation concerns and strict government policy regulation about such organisms. Therefore, one of the main challenges in using the CRISPR/Cas system in commercial agriculture is to improve the plant’s agronomic traits by genome editing, meanwhile obtaining transgene-free mutant plants with stable transmissible properties. DNA-free genome editing technology in plants is new and began in 2015, and since then great advances have been made on producing transgenic-free genome-edited plants. The exogenous CRISPR/Cas cassette and other transgenes could be eliminated by multiple approaches after the target gene has been edited. For example, the CRISPR/Cas expression cassette can be removed through genetic segregation for plants that are reproduced sexually, and the end products are CRISPR-edited but transgene-free. Transgene-free gene-edited plants may also be generated by non-transgenic approaches, such as transient expression of mRNA encoding Cas nuclease and guide RNA without DNA integration, preassembled ribonucleoprotein and sgRNA complex transfection, fluorescence marker-assisted transgene cassette selection and elimination, virus-mediated and nano-material mediated genome editing technologies. By using these methods, people can obtain the genome-edited end plant product with increased agronomic traits, while no additional foreign DNA fragments have been introduced into the plant genome. Until now, at least 14 transgene-free and genome-edited plant species have been generated using this technology. In this review, we have highlighted these achievements, including methods, applications and the possibilities of their commercialization in plant breeding. We also discuss the current problems in using this technology, such as very low efficiency, limited gene modification types and labor-consuming aspects. With the development of new plant cell transformation methods and the applications of new typical CRISPR/Cas systems, the potential strategies for improving this revolutionary technology in the future are also discussed.

Background: The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA-despite being successful in E. coli and animal models-still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. Methods: Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a ‘gene of interest’. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. Results: The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. Conclusion: We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications. National Research Foundation (NRF) Singapore-MIT Alliance for Research and Technology (SMART) Published version This work was supported through grants from the Singapore-MIT Alliance for Research and Technology, Grant Award Numbers M4062347.080 and M4062198.080 to H.M., and the National Research Foundation (NRF)Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF-CRP21-2018-0002 to P.D. Funding for open access charge: National Research Foundation Competitive Research Programme, Singapore (NRF-CRP21-2018-0002).

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the alpha-galactosidase A (GLA) gene, which encodes the exogalactosyl hydrolase, alpha-galactosidase A (α-Gal A). Deficient α-Gal A activity results in the progressive, systemic accumulation of its substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), leading to renal, cardiac, and/or cerebrovascular disease and early demise. The current standard treatment for Fabry disease is enzyme replacement therapy, which necessitates lifelong biweekly infusions of recombinant enzyme. A more long-lasting treatment would benefit Fabry patients. Here, a gene therapy approach using an episomal adeno-associated viral 2/6 (AAV2/6) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette was evaluated in a Fabry mouse model that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. A detailed 3-month pharmacology and toxicology study showed that administration of a clinical-scale-manufactured AAV2/6 vector resulted in markedly increased plasma and tissue α-Gal A activities, and essentially normalized Gb3 and Lyso-Gb3 at key sites of pathology. Further optimization of vector design identified the clinical lead vector, ST-920, which produced several-fold higher plasma and tissue α-Gal A activity levels with a good safety profile. Together, these studies provide the basis for the clinical development of ST-920., Graphical Abstract

Non-viral gene delivery into the liver generally mediates a transient transgene expression. A comparative analysis was performed using two gene vectors, pFAR4 and pKAR4, which differ by the absence or presence of an antibiotic resistance marker, respectively. Both plasmids carried the same eukaryotic expression cassette composed of a sulfamidase (Sgsh) cDNA expressed from the human alpha antitrypsin liver-specific promoter. Hydrodynamic injection of the pFAR4 construct resulted in prolonged sulfamidase secretion from the liver, whereas delivery of the pKAR4 construct led to a sharp decrease in circulating enzyme. After induction of hepatocyte division, a rapid decline of sulfamidase expression occurred, indicating that the pFAR4 derivative was mostly episomal. Quantification analyses revealed that both plasmids were present at similar copy numbers, whereas Sgsh transcript levels remained high only in mice infused with the pFAR4 construct. Using a chromatin immunoprecipitation assay, it was established that the 5′ end of the expression cassette carried by pKAR4 exhibited a 7.9-fold higher heterochromatin-to-euchromatin ratio than the pFAR4 construct, whereas a bisulfite treatment did not highlight any obvious differences in the methylation status of the two plasmids. Thus, by preventing transgene expression silencing, the pFAR4 gene vector allows a sustained transgene product secretion from the liver., Graphical Abstract, Non-viral gene delivery into the liver generally mediates a transient transgene expression. Marie and colleagues developed an antibiotic-free gene vector that mediates prolonged protein secretion from the liver by preventing transcriptional silencing and heterochromatin formation.

By using the more effective IRES (internal ribosome entry site) element, an innovative cap‐independent protein expression system mediated by RNA polymerase I, which only transcribes rRNA normally, was established in Saccharomyces cerevisiae. Such system made the reporter gene yEGFP3 express variation especially after mutagenesis. Combined with flow cytometry, cells with high fluorescence intensity were sorted from 200,000 cells and the strain with 58% RNA content higher than the parental strain was obtained. So the model for large‐scale screening of high‐nucleic‐acid content yeasts was set up., Summary Saccharomyces cerevisiae is the preferred source of RNA derivatives, which are widely used as supplements for foods and pharmaceuticals. As the most abundant RNAs, the ribosomal RNAs (rRNAs) transcribed by RNA polymerase I (Pol I) have no 5′ caps, thus cannot be translated to proteins. To screen high‐nucleic‐acid content yeasts more efficiently, a cap‐independent protein expression system mediated by Pol I has been designed and established to monitor the regulatory changes of rRNA synthesis by observing the variation in the reporter genes expression. The elements including Pol I‐recognized rDNA promoter, the internal ribosome entry site from cricket paralytic virus which can recruit ribosomes internally, reporter genes (URA3 and yEGFP3), oligo‐dT and an rDNA terminator were ligated to a yeast episomal plasmid. This system based on the URA3 gene worked well by observing the growth phenotype and did not require the disruption of cap‐dependent initiation factors. The fluorescence intensity of strains expressing the yEGFP3 gene increased and drifted after mutagenesis. Combined with flow cytometry, cells with higher GFP level were sorted out. A strain showed 58% improvement in RNA content and exhibited no sequence alteration in the whole expression cassette introduced. This study provides a novel strategy for breeding high‐nucleic‐acid content yeasts.

Actinidia arguta and related species produce small edible kiwifruit (kiwiberry) with attractive consumer features and high nutritional value. As a recently developed crop, kiwiberry can be improved further by classical breeding or genetic manipulation. The currently available Agrobacterium-mediated transformation protocol is only applicable to a limited number of kiwiberry genotypes such as the female cultivar ‘Hortgem Tahi’. We developed protocols for regeneration and Agrobacterium-mediated transformation of two A. arguta genotypes, female AA06-01 and male AA05-06, also applicable to ‘Hortgem Tahi’. Altered composition of basal salts in the callus-induction media was sufficient to prevent callus browning in AA06-01 and ‘Hortgem Tahi’, but not in AA05-06. Altering the hormone composition to avoid a prolonged callus stage was successfully used to prevent browning and induce shoot development in all three genotypes. Using our improved protocols, Agrobacterium-mediated transformation of a binary vector carrying the neomycin phosphotransferase II (nptII) expression cassette gave rise to kanamycin-resistant plants of both AA06-01 and AA05-06 and the presence of the nptII transgene was confirmed by genomic PCR. The improved protocols were also used to ectopically overexpress the anthocyanin-related transcription factor AcMYB110 in ‘Hortgem Tahi’ giving rise to purple callus with elevated anthocyanin accumulation. AcMYB110 expression and increased levels of anthocyanins were detected in mature leaves of established transgenic plants compared to controls, clearly demonstrating the important role for MYB110 in regulation of anthocyanin accumulation in kiwiberry. The protocols developed during this study provide tools for further functional analyses and genetic manipulation of kiwiberry genotypes. Using a newly developed transformation method we were able to generate transgenic lines of three Actinidia arguta genotypes and demonstrate an important role for MYB110 in anthocyanin accumulation in kiwiberry.

Background. A selective system based on the M1 virus of the yeast Saccharomyces cerevisiae was proposed. Methods. To create a recipient strain, a DNA fragment encoding the killer toxin of the M1 virus under the control of the regulated promoter of the GAL1 gene was inserted into the genome of S. cerevisiae strains Y-1236 and Y-2177. Results. Integration of such expression cassette leads to the conditional lethality resulting strains die on a medium with galactose when killer toxin synthesis occurs. A linear DNA fragment containing the gene of interest flanked by sequences homologous to the promoter of the GAL1 gene and the termination region of the CYC1 gene is used to transform the obtained strains. During transformation due to homologous recombination, the sequence encoding the killer toxin is cleaved and the transformants grow on a medium with galactose. Conclusion. The proposed selective system combines the main advantages of other systems: the use of simple media, without the need to add expensive antibiotics, and a simplified technique for constructing expression cassettes and selecting transformants.

As the main immunogen that could stimulate neutralized antibody in pigs, recombinant E2 protein of CSFV was expressed in CHO‐dhfr−cells driven by endogenous Txnip promoter from Chinese hamster. Different fragments of Txnip promoter were amplified by PCR from isolated genomic DNA of CHO cells and cloned into different expression vectors. Compared with CMV promoter, CHO‐pTxnip‐4‐rE2 (F12) cell clone with the highest yield of rE2 protein was established by random insertion of the expression cassette driven by 860 bp sequences of Txnip promoter. In combination with treatment of 800 nM MTX for copy amplification of inserted expression cassette, the dynamic expression profile of rE2 protein was observed. Then inducible expression strategy of balance between viable cell density and product yield was conducted by mixed addition of 0.1 mM NADH and 0.1 mM ATP in culture medium at day 3 of batch‐wise culture. It could be concluded that Txnip promoter would be a promising alternative promoter for recombinant antigen protein expression in transgenic cells.

Most expression systems are tailored for model organisms rather than nonmodel organisms. However, heterologous gene expression in model organisms constrains the innate advantages of original strain carrying gene of interest. In this study, T7 expression system was developed in nonmodel bacterium Klebsiella pneumoniae for production of chemicals. First, we engineered a recombinant K. pneumoniae strain harboring two vectors. One vector was used to express T7 RNA polymerase (T7 RNAP) which would drive the expression of egfp in the other vector. This recombinant strain demonstrated 15.73-fold of fluorescence relative to wild-type K. pneumoniae and showed similar level of fluorescence to recombinant Escherichia coli overexpressing egfp. When egfp was replaced by puuC, an endogenous aldehyde dehydrogenase catalyzing 3-hydroxypropionic acid (3-HP) biosynthesis in K. pneumoniae, the recombinant strain coexpressing T7 RNAP and PuuC showed high-level PuuC expression. In shake-flask cultivation, this recombinant strain produced 1.72 g/L 3-HP in 24 hr, which was 3.24 times that of wild-type K. pneumoniae (0.53 g/L). To mitigate plasmid burden, the vector expressing T7 RNAP was eliminated, but the T7 RNAP expression cassette was integrated into K. pneumoniae genome. The resulting strain harboring only PuuC expression vector produced 2.44 g/L 3-HP in 24 hr under shake-flask conditions, which was 1.46 times that of the strain harboring both T7 RNAP and PuuC expression vectors. In bioreactor cultivation, this strain generated 67.59 g/L 3-HP and did not show significantly halted growth. Overall, these results indicate that the engineered T7 expression system functioned efficiently in K. pneumoniae. This study provides a paradigm for the development of T7 expression system in prokaryotes.

Systemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3′ untranslated region (3′ UTR) of the expression cassette. Here, we aimed to generate such miRNA-122- and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1). We successfully validated the vector functionality in murine cell lines corresponding to tissues targeted by AAV9. Next, we evaluated biodistribution of transgene expression following systemic vector delivery to HO-1-deficient mice of mixed C57BL/6J × FVB genetic background. Although AAV genomes were present in the hearts of these animals, HO-1 protein expression was either absent or significantly impaired. We found that miRNA-122, earlier described as liver specific, was present also in the hearts of C57BL/6J × FVB mice. Various levels of miRNA-122 expression were observed in the hearts of other mouse strains, in heart tissues of patients with cardiomyopathy, and in human induced pluripotent stem cell-derived cardiomyocytes in which we also confirmed such posttranscriptional regulation of transgene expression. Our data clearly indicate that therapeutic utilization of miRNA-based regulation strategy needs to consider inter-individual variability., Graphical Abstract, Kraszewska et al. investigated miRNA-122- and miRNA-206-mediated posttranscriptional regulation to restrict AAV9-encoded transgene expression to mouse cardiac tissue. A low level of miRNA-122 present in the investigated murine heart, as well as in human cardiomyocytes, considerably decreased transgene expression, indicating that the use of miRNA target sites should be carefully considered.

The use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltppro), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as “altruistic transformation.”

Human epidermal growth factor (hEGF) is a crucial factor in wound healing, and therefore has wide applications in the pharmaceutical and cosmetics industries. To improve the production of hEGF protein for the industrial scale, we established an hEGF gene construct for recombinant secretion by Bacillus subtilis. Examination of the secretion abilities of various signal peptides (SPs) revealed six SPs that could effectively guide hEGF secretion from B. subtilis into the culture medium. The highest production level was obtained when the hEGF gene was fused with the xynD SP (lipo type). Two copies of the hEGF expression cassette had the same effect on improving production yield as isopropyl-β- d -1-thiogalactopyranoside (IPTG) induction, and the highest yield of hEGF can reach 360 ± 9.41 mg/L. Moreover, high-purity hEGF was obtained using a His-tagged purification system. Overall, these results demonstrate that B. subtilis is a suitable cell factory for hEGF production with improved yield compared to previous systems.

Commonly used and well accepted approaches are lacking for site-directed modification of adenoviral vectors. Here, we attempt to introduce an easy-to-implement strategy for such purpose with an example of establishing a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR products of GFP expression cassette and plasmid backbone were fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA assembly. NdeI-digested fragment of pMDXE3GA was brought back to pKAd5 to form the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension K562 cells were investigated. Expression of target gene was significantly enhanced in both cell lines infected with HAdV5-XE3GA due to virus replication. However, propagation of virus could not sustain in culture of K562 cells. Shuttle plasmid pSh5RC-GFP was constructed to facilitate exchange of transgene. In summary, the strategy of combined DNA assembly and restriction-ligation cloning is functional, cost-effective and suitable for genetic modification of adenovirus.

Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.