Author Correction: A streamlined base editor engineering strategy to reduce bystander editing.

Cell-derived extracellular vesicles for CRISPR/Cas9 delivery: engineering strategies for cargo packaging and loading.

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

EDITORIAL article Front. Microbiol., 11 January 2016Sec. Microbial Physiology and Metabolism Volume 6 - 2015 | https://doi.org/10.3389/fmicb.2015.01562

Rapid identification of pathogens responsible for bloodstream infection is critical for early intervention and effective treatment. Reporter phages, which are known for their exceptional sensitivity and specificity in pathogen detection, have garnered significant interest. In this study, we systematically evaluate phage genome editing strategies that combine homologous recombination with the CRISPR-Cas9 system. We investigate the impacts of homologous arm length, sgRNA activity, target site, and plasmid interactions on editing efficiency. Our results demonstrate that successful genome editing depends on both sufficient cleavage pressure and optimal homologous arm length, particularly when using low-activity sgRNAs. On the basis of these findings, we develop a highly efficient gene editing strategy TPMSR (triple-plasmid-mediated synchronous recombination) that overcomes the limitations of conventional methods that rely on high-activity sgRNA and restricted editing sites. Using the TPMSR strategy, we integrate the Nluc gene into phage T7, generating the reporter phage T7:: Nluc, which is then incorporated into a microfluidic chip. Validation with 51 clinical isolates demonstrates outstanding sensitivity, specificity, and accuracy in detecting E. coli in blood within 1.5 h at concentrations less than 30 CFU/mL. This study presents a robust strategy for phage genome engineering and develops a promising method for the rapid diagnosis of bloodstream infections caused by Escherichia coli.

Plant genetic engineering, a recent technological advancement in the field of plant science, is an important tool used to improve crop quality and yield, to enhance secondary metabolite content in medicinal plants or to develop crops for sustainable agriculture. A new approach based on nanoparticle-mediated gene transformation can overcome the obstacle of the plant cell wall and accurately transfer DNA or RNA into plants to produce transient or stable transformation. In this review, several nanoparticle-based approaches are discussed, taking into account recent advances and challenges to hint at potential applications of these approaches in transgenic plant improvement programs. This review also highlights challenges in implementing the nanoparticle-based approaches used in plant genetic engineering. A new technology that improves gene transformation efficiency and overcomes difficulties in plant regeneration has been established and will be used for the denovo production of transgenic plants, and CRISPR/Cas9 genome editing has accelerated crop improvement. Therefore, we outline future perspectives based on combinations of genome editing, nanoparticle-mediated gene transformation and denovo regeneration technologies to accelerate crop improvement. The information provided here will assist an effective exploration of the technological advances in plant genetic engineering to support plant breeding and important crop improvement programs.

Targeted transcriptome engineering, in contrast to genome engineering, offers a complementary and potentially tunable and reversible strategy for cellular engineering. In this regard, adenosine to inosine (A-to-I) RNA base editing was recently engineered to make programmable base conversions on target RNAs. Similar to the DNA base editing technology, A-to-I RNA editing may offer an attractive alternative in a therapeutic setting, especially for the correction of point mutations. This Perspective introduces five currently characterized RNA editing systems and serves as a reader's guide for implementing an appropriate RNA editing strategy for applications in research or therapeutics.

Adoptive cell transfer (ACT) of engineered T cells is effective against B cell malignancies but has faltered against solid tumors due to the immunosuppressive tumor microenvironment (TME). FAS ligand (FASL) and transforming growth factor β (TGF-β) are key mediators of T cell dysfunction in the TME and overexpressing dominant negative (dn) forms of their receptors in T cells increases anti-tumor efficacy in solid tumor models. However, an approach that directly targets the endogenous genes would be more amenable to multiplex editing and reduce competition with WT alleles. Here, we employ base editing (BE) in primary human T cells to install naturally occurring dominant negative FAS and TGFβR2 mutations. In vitro survival and proliferation assays demonstrate that BE T cells are resistant to pro-apoptotic and anti-proliferative effects of FAS and TGF-β signaling. Chimeric antigen receptor (CAR)-T cells with BE-installed dn TGFβR2 or dn FAS exhibit improvements in cytotoxicity, while dn TGFβR2 CAR T demonstrate increased persistence and reduced expression of phenotypic markers of exhaustion compared to controls. Moreover, BE-engineered dn CAR T outperform lentiviral-engineered cDNA over expression counterparts in several functional assays. Considering the efficiency of BE and its amenability for multiplex editing, our approach lends itself to engineering strategies necessary to overcome T cell dysfunction in solid tumors.

CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.

A multitude of climatic factors, such as temperature, drought, salinity, and heavy metal toxicity, affect agricultural production. In this respect, to avert a drop in agricultural productivity resulting from climate change, plant breeding innovations and genetic engineering strategies to boost abiotic stress tolerance are important. Biotechnologists have been able to tweak any DNA sequence with excellent accuracy via the latest discoveries in first-generation genome editing techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). These strategies, however, are expensive and laborious, since they require complex steps involving protein engineering. Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas strategy as a second-generation genome editing system has gained great interest over the last few years as an effective method to create heritable genetic mutations. Using the CRISPR/Cas strategy, a handful of studies have been reported to date on improving abiotic stress resilience in crop plants. In addition, the development of more specialized forms of Cas9 proteins such as Cpf1, dCas9 greatly enhances the flexibility of the CRISPR/Cas system. This chapter focuses on the perspective and promising applications, recent advances, and potential challenges of targeted genome engineering for increased plant tolerance to abiotic stress and productivity.

Synthetic biology and metabolic engineering strategies in identifying and producing plant natural products; with emphasis on the CRISPR/Cas systems

CAR T-cell therapy has become a transformative modality in oncology, demonstrating sustained clinical efficacy in hematologic malignancies. Recent investigations have expanded its potential beyond cancer to immune-mediated disorders, including autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus, as well as chronic viral infections including HIV and hepatitis B. This review examines the mechanistic foundations of CAR-T cells, advances in universal and allogeneic engineering strategies designed to mitigate graft-versus-host disease and host rejection, and emerging in vivo gene-delivery platforms that aim to bypass conventional ex vivo manufacturing. We further evaluate safety-control architectures, including logic-gated and inducible systems, and discuss translational barriers related to scalability, manufacturing standardization, and long-term immune durability. While technological innovations in genome editing, synthetic biology, and computational design continue to refine CAR-T platforms, substantial biological and logistical challenges remain. A critical synthesis of these evolving strategies is necessary to distinguish incremental optimization from paradigm-shifting advances and to define the future trajectory of CAR-based immunotherapy across oncology and immune-mediated diseases.

Many metabolic pathways in bacteria are modulated by metabolite-sensing riboswitches, which regulate gene expression at the level of transcription elongation or translation initiation. Riboswitches represent promising targets to modulate expression of genes and operons relevant for the biotechnological production of commercially relevant compounds. In Firmicutes, approximately 70% of all putative and validated riboswitches (are predicted to) act exclusively at the transcriptional level using a termination-antitermination mechanism. In a first attempt to interfere with purine-sensing riboswitches and deregulate purine metabolism in Bacillus subtilis, a set of synthetic small RNAs (sRNAs) targeting the purine-sensing aptamers were designed to impair ligand binding using rational design combined with in silico evolution. However, the designed sRNAs did not show any activity in vivo on the riboswitch controlling purine biosynthesis (pur operon riboswitch). The effect of the antisense RNA (asRNA) perfectly complementary to the aptamer of the pur operon riboswitch was also tested; The asRNA did not affect negatively expression of a riboswitch-regulated lacZ gene, yet similarly to the partially complementary sRNAs, the asRNA did not impair the downregulation exerted by the riboswitch in the presence of ligand. Finally, expression of the small RNAs in B. subtiliswas quantified, and the kinetic limitations for their hybridization with the aptamer and their competition with the ligand are discussed. A second metabolic engineering strategy based on editing the genome of B. subtilis with regard to transcriptional riboswitches was investigated. Removal of the riboswitches that control purine biosynthesis and riboflavin biosynthesis in B. subtilis led to auxotrophic strains. As an alternative, a rational approach was developed for engineering transcriptional riboswitches independently from the availability of their 3D structures. This approach consists in the identification and deletion of a key nucleotide sequence exclusively involved in transcription termination without affecting formation of other secondary and tertiary structures potentially involved in other roles. To demonstrate the efficacy of the approach, it was applied to derepress the purine and the riboflavin biosynthetic pathways in B. subtilis. Following the proof of concept using specialized reporter strains, the approach was implemented into a B. subtilis wild-type strain employing CRISPR-Cas genome editing. The CRISPRCas9 system displayed an efficiency of 61% in editing the genome, and the resulting purine and riboflavin production strains were characterized at the level of gene expression, metabolite synthesis, and growth. With a substantial enhancement observed at each level, the strategy established here represents a powerful tool for deregulating pathways modulated by transcriptional riboswitches. Finally, applying this strategy to derepress the purine pathway of an industrial riboflavin overproducing strain, with impaired growth, led to an increase in biomass by 53% and resulted in an enhanced total production of riboflavin in the culture.

Elevated sea surface temperatures from a severe and prolonged El Niño event (2014–2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.

Innovative genome editing technologies are accumulating and are rapidly elucidating the genetic basis of many cell functions. However, many disease phenotypes and physiological processes are governed by complex networks involving many gene products. Thus, numerous phenotypes need to be unraveled using multiplex genome editing techniques and in silico analysis. Furthermore, these approaches must further leverage legacy knowledge to identify the genes involved and understand the cell response. As an example, we unraveled the core cellular network controlling the Warburg effect, also known as aerobic glycolysis. This effect involves the tendency of rapidly‐proliferating cells to consume excess glucose and secrete copious amounts of lactic acid, despite having adequate oxygen for more efficient oxidative phosphorylation. This response is a hallmark of cancer, immune cell expansion, and other processes with rapidly proliferating cells. However, efforts to test the purpose of Warburg metabolism have been stymied by the difficulty to generate cell lines that are unable to produce lactic acid, since genes involved have proven to be essential or synthetic lethal in immortalized cells. We discovered a genetic circuit involving multiple genes that control lactic acid secretion, and using a multiplex genome editing with CRISPR, we successfully eliminated lactic acid secretion and enabled the deletion of multiple "essential" genes. Surprisingly, the cells show improved metabolic and growth phenotypes, despite the elimination of this fundamental metabolic activity. To understand how immortalized mammalian cells can cope without this seemingly essential metabolic process, we conducted a comprehensive analysis of these cell lines using time‐course RNA‐Seq, metabolomics, and analysis with a genome‐scale metabolic network model we have developed for Chinese hamster ovary cells1. Thus, through a multiplex metabolic engineering effort and comprehensive systems biology analysis, we have been able to engineer out a hallmark phenotype of proliferating cells and begin to understand now a cell can survive without a seemingly essential process.Support or Funding InformationThe Novo Nordisk Foundation Center for Biosustainability (NNF10CC1016517)

The explosion of interest in CRISPR-Cas systems over recent years has delivered rapid advances in precision genome engineering technologies that hold enormous potential for application across the biotechnology sector. Within the cell therapy space, adoptive immunotherapies continue to lead the translation of increasingly sophisticated genetic engineering strategies toward clinical application. Although early focus was placed on engineering primary immune cells collected from patient blood, the democratization of these therapeutics will require the development of off-the-shelf allogeneic cell therapy products that can be manufactured at scale. Pluripotent stem cells (PSCs) represent an attractive substrate for such products, and many of the challenges that must be overcome to realize the application of PSCs in adoptive cell therapies are common with those holding back regenerative cell-replacement therapies. This article discusses how genome editing can overcome these challenges and highlights the potential of rapidly advancing second-generation technologies in the development of novel cell-based therapeutics.