The new frontier in CHO cell line development: From random to targeted transgene integration technologies.

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

pIGLET: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish.

Standard methods for transgenesis in zebrafish depend on random transgene integration into the genome followed by resource-intensive screening and validation. Targeted vector integration into validated genomic loci using phiC31 integrase-based attP/attB recombination has transformed mouse and Drosophila transgenesis. However, while the phiC31 system functions in zebrafish, validated loci carrying attP-based landing or safe harbor sites suitable for universal transgenesis applications in zebrafish have not been established. Here, using CRISPR-Cas9, we converted two well-validated single insertion Tol2-based zebrafish transgenes with long-standing genetic stability into two attP landing sites, called phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET). Generating fluorescent reporters, loxP-based Switch lines, CreERT2 drivers, and gene-regulatory variant reporters in the pIGLET14a and pIGLET24b landing site alleles, we document their suitability for transgenesis applications across cell types and developmental stages. For both landing sites, we routinely achieve 25-50% germline transmission of targeted transgene integrations, drastically reducing the number of required animals and necessary resources to generate individual transgenic lines. We document that phiC31 integrase-based transgenesis into pIGLET14a and pIGLET24b reproducibly results in representative reporter expression patterns in injected F0 zebrafish embryos suitable for enhancer discovery and qualitative and quantitative comparison of gene-regulatory element variants. Taken together, our new phiC31 integrase-based transgene landing sites establish reproducible, targeted zebrafish transgenesis for numerous applications while greatly reducing the workload of generating new transgenic zebrafish lines.

Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

LIFE-Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms.

Molecular characterization of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion and tandem repeats. Herein, we developed a universal Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (LIFE-Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE-Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging ~6275 bp were enriched and sequenced, generating ~26 352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with next-generation whole-genome sequencing, LIFE-Seq achieved better data integrity and accuracy, greater universality and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE-Seq could be applied in molecular characterization of transgenic crops and animals, and complex DNA structure analysis in genetics research.

Transgene-free Genome Editing in Plants.

Genome editing is widely used across plant species to generate and study the impact of functional mutations in crop improvement. However, transgene integration in plant genomes raises important legislative concerns regarding genetically modified organisms. Several strategies have been developed to remove or prevent the integration of gene editor constructs, which can be divided into three major categories: 1) elimination of transgenic sequences via genetic segregation; 2) transient editor expression from DNA vectors; and 3) DNA-independent editor delivery, including RNA or preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs). Here, we summarize the main strategies employed to date and discuss the advantages and disadvantages of using these different tools. We hope that our work can provide important information concerning the value of alternative genome editing strategies to advance crop breeding.

Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system.

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.

Linking Chromosomal Silencing With Xist Expression From Autosomal Integrated Transgenes.

Xist is the master regulator of X-Chromosome Inactivation (XCI), the mammalian dosage compensation mechanism that silences one of the two X chromosomes in a female cell. XCI is established during early embryonic development. Xist transgene (Tg) integrated into an autosome can induce transcriptional silencing of flanking genes; however, the effect and mechanism of Xist RNA on autosomal sequence silencing remain elusive. In this study, we investigate an autosomal integration of Xist Tg that is compatible with mouse viability but causes male sterility in homozygous transgenic mice. We observed ectopic Xist expression in the transgenic male cells along with a transcriptional reduction of genes clustered in four segments on the mouse chromosome 1 (Chr 1). RNA/DNA Fluorescent in situ Hybridization (FISH) and chromosome painting confirmed that Xist Tg is associated with chromosome 1. To determine the spreading mechanism of autosomal silencing induced by Xist Tg on Chr 1, we analyzed the positions of the transcriptionally repressed chromosomal sequences relative to the Xist Tg location inside the cell nucleus. Our results show that the transcriptionally repressed chromosomal segments are closely proximal to Xist Tg in the three-dimensional nucleus space. Our findings therefore support a model that Xist directs and maintains long-range transcriptional silencing facilitated by the three-dimensional chromosome organization.

Identification of transgene integration site and anatomical properties of fluorescence intensity in a EGFP transgenic chicken line.

Transgenic birds are commonly used for time-lapse imaging and fate mapping studies in developmental biology. When researchers use transgenic birds expressing fluorescent protein, they need to understand the integration site of the transgene in the genome and the intensity of fluorescence in the tissues of interest. In this study, we determined the integration site of the transgene and fluorescence property of developing organs in our transgenic chicken line generated by lentivirus infection. The transgene was localized between exons 3 and 4 of MED27. Some homozygotes and heterozygotes appeared to be lethal at early embryonic stages. We performed histological analysis of EGFP expression in transgenic embryos at St. 14, 17, and 24 by immunohistochemistry with anti-GFP antibody on paraffin sections. Next, we cut cryosections and quantified direct EGFP intensity from the transgene in each tissue without performing immunohistochemistry. These results revealed that EGFP intensity in each tissue was unique in developing embryos and changed according to developmental stages. Finally, we demonstrated that EGFP-expressing cells in a micromass culture with co-culturing wild-type cells were clearly distinguishable via live cell imaging. These results provide essential information on the potential of our transgenic line and indicate that these transgenic chicken lines are useful for research associated with developmental biology.

Mitigating Clonal Variation in Recombinant Mammalian Cell Lines.

Mammalian expression platforms are primary production systems for therapeutic proteins that require complex post-translational modifications. Current processes used for developing recombinant mammalian cell lines generate clonal cell lines with high phenotypic heterogeneity, which has puzzled researchers that use mammalian cell culture systems for a long time. Advances in mammalian genome-editing technologies and systems biotechnology have shed light on clonal variation and enabled rational cell engineering in a targeted manner. We propose a new approach for a next-generation cell line development platform that can minimize clonal variation. Combined with the knowledge-based selection of ideal integration sites and engineering targets, targeted integration-based cell line development will allow tailored control of recombinant gene expression with predicted phenotypes.

Addressing concerns over the fate of DNA derived from genetically modified food in the human body: A review.

Global commercialization of GM food and feed has stimulated much debate over the fate of GM food-derived DNA in the body of the consumer and as to whether it poses any health risks. We reviewed the fate of DNA derived from GM food in the human body. During mechanical/chemical processing, integrity of DNA is compromised. Food-DNA can survive harsh processing and digestive conditions with fragments up to a few hundred bp detectable in the gastrointestinal tract. Compelling evidence supported the presence of food (also GM food) derived DNA in the blood and tissues of human/animal. There is limited evidence of food-born DNA integrating into the genome of the consumer and of horizontal transfer of GM crop DNA into gut-bacteria. We find no evidence that transgenes in GM crop-derived foods have a greater propensity for uptake and integration than the host DNA of the plant-food. We found no evidence of plant-food DNA function/expression following transfer to either the gut-bacteria or somatic cells. Strong evidence suggested that plant-food-miRNAs can survive digestion, enter the body and affect gene expression patterns. We envisage that this multi-dimensional review will address questions regarding the fate of GM food-derived DNA and gene-regulatory-RNA in the human body.

Integrated Multimodal Evaluation of Genotoxicity in ZFN-Modified Primary Human Cells.

Iatrogenic adverse events in clinical trials of retroviral vector-mediated gene-corrected cells have prioritized the urgent need for more comprehensive and stringent assessment of potentially genotoxic off-target alterations and the biosafety of cells intended for therapeutic applications. Genome editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 nuclease systems are being investigated as safer and efficient alternatives for site-directed genome modification. Using site-specific integration into the AAVS1 locus of primary human cells as an example, we present an integrated approach to multimodal investigation of off-target alterations and an evaluation of potential genotoxicity induced by ZFN-mediated integration of a therapeutic transgene.

Rapid Integration of Multi-copy Transgenes Using Optogenetic Mutagenesis in Caenorhabditis elegans.

Stably transmitted transgenes are indispensable for labeling cellular components and manipulating cellular functions. In Caenorhabditis elegans, transgenes are generally generated as inheritable multi-copy extrachromosomal arrays, which can be stabilized in the genome through a mutagenesis-mediated integration process. Standard methods to integrate extrachromosomal arrays primarily use protocols involving ultraviolet light plus trimethylpsoralen or gamma- or X-ray irradiation, which are laborious and time-consuming. Here, we describe a one-step integration method, following germline-mutagenesis induced by mini Singlet Oxygen Generator (miniSOG). Upon blue light treatment, miniSOG tagged to histone (Histone-miniSOG) generates reactive oxygen species (ROS) and induces heritable mutations, including DNA double-stranded breaks. We demonstrate that we can bypass the need to first establish extrachromosomal transgenic lines by coupling microinjection of desired plasmids with blue light illumination on Histone-miniSOG worms to obtain integrants in the F3 progeny. We consistently obtained more than one integrant from 12 injected animals in two weeks. This optogenetic approach significantly reduces the amount of time and labor for transgene integration. Moreover, it enables to generate stably expressed transgenes that cause toxicity in animal growth.

Inhibitory neuron-specific Cre-dependent red fluorescent labeling using VGAT BAC-based transgenic mouse lines with identified transgene integration sites.

Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.

Homology-Independent Integration of Plasmid DNA into the Zebrafish Genome.

Targeting nucleases like zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system have revolutionized genome-editing possibilities in many model organisms. They allow the generation of loss-of-function alleles by the introduction of double-strand breaks at defined sites within genes, but also more sophisticated genome-editing approaches have become possible. These include the integration of donor plasmid DNA into the genome by homology-independent repair mechanisms after CRISPR/Cas9-mediated cleavage. Here we present a protocol outlining the most important steps to target a genomic site and to integrate a donor plasmid at this defined locus.

Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence.

Genome editing in plants via designed zinc finger nucleases.

The ability to create DNA double-strand breaks (DSBs) at specified genomic locations, which then stimulate the cell's naturally occurring DNA repair processes, has introduced intriguing possibilities for genetic modification. Zinc finger nucleases (ZFNs) are designed restriction enzymes consisting of a nonspecific cleavage domain fused to sequence-specific DNA binding domains. ZFN-mediated DSB formation at endogenous genomic loci followed by error-prone non-homologous end joining (NHEJ) repair can result in gene-specific mutations via nucleotide base pair insertions or deletions. Similarly, specific DNA sequence modifications can be made by providing donor DNA templates homologous to sequences flanking the cleavage site via homology-directed repair (HDR). Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs. Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR. Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants. As with conventional plant transformation technology, the efficiency of genome editing is absolutely dependent on the ability to initiate, maintain, and regenerate plant cell and tissue cultures.