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Gene-editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene-editing-based therapy is unlikely to be successful; therefore, a multi-gene knockout strategy is preferred for effective GBM inhibition. Here, a non-invasive, biodegradable brain-targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half-life, efficient blood-brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient-derived GSC xenograft mouse models with negligible off-target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi-oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM.

The past decades have seen increasing calls to actively involve publics in the governance of science and technology. Many public engagement initiatives aim to facilitate the formation of public opinion. But what is an opinion? While the notion is often taken as self-evident, different imaginaries of what opinions are and how they should be formed are highly consequential for shaping relations between technoscience and society. Based on participant observations and interviews, we analyze how "opinion" is enacted as an emergent object and category with specific properties and uses in a series of public engagement events on genome editing. By identifying two prevalent goals tied to partially conflicting imaginaries of opinion—open deliberation and "capturing" public opinion—our analysis contributes to a more reflective understanding of the tensions that participation facilitators navigate when making opinions their central points of intervention in the coevolving relationship between technologies and their publics. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) have emerged as one of the most therapeutically significant adult stem cells, paving way for a range of novel curative regimens over decades. HSCs are transplanted, either directly or post restorative genetic engineering in order to repopulate a healthy hematopoietic homeostasis in patients with disorders affecting the blood and immune cells. Despite being an extensively studied system, the maintenance and expansion of functional HSCs ex vivo remains a major bottleneck. The challenge primarily stems from difficulties in reproducing HSC self-renewal divisions and gradual depletion of stemness characters, in vitro. Refining the in vitro culture can be particularly beneficial in the case of cord blood HSCs (CB-HSCs), as inadequate numbers in a single umbilical cord limits its therapeutic potential. In recent years, molecular dissection of HSC stemness has significantly improved in vitro hematopoietic stem and progenitor cells (HSPCs) culture. Despite such significant progress, lacunae exist in fully understanding all the underlying mechanisms and their interplay active in bona fide HSCs, and how it transforms when cells proliferate in culture. A new groundbreaking study titled "MYCT1 controls environmental sensing in human haematopoietic stem cells", published in Nature in June 2024, sheds light on this complex field. Through a series of experiments, including knock-down, overexpression, single-cell RNA sequencing, and transplantation, the study identifies a previously unknown role of the MYC target 1 (MYCT1) protein in HSC maintenance. This protein acts as a crucial regulator of human HSCs, with high expression in primitive HSCs and subsequently downregulated during ex vivo culture. The study reveals that MYCT1 plays a vital role in moderating endocytosis and environmental sensing in HSCs, processes thereby essential for maintaining HSC stemness and function. This commentary will discuss the implications of the new findings for cord blood expansion in cell therapies and HSPC culture for gene therapy applications, providing valuable insights for the field of hematopoietic regenerative medicine. [ABSTRACT FROM AUTHOR]

Summary: The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase‐like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C4 photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell‐level electron fluxes remained unknown.We have created gene‐edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells.We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO2.Our results indicate that NDH‐mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO2 fixation by the C3 cycle in BS cells. [ABSTRACT FROM AUTHOR]

Abstract Hematopoietic stem cells (HSCs) have emerged as one of the most therapeutically significant adult stem cells, paving way for a range of novel curative regimens over decades. HSCs are transplanted, either directly or post restorative genetic engineering in order to repopulate a healthy hematopoietic homeostasis in patients with disorders affecting the blood and immune cells. Despite being an extensively studied system, the maintenance and expansion of functional HSCs ex vivo remains a major bottleneck. The challenge primarily stems from difficulties in reproducing HSC self-renewal divisions and gradual depletion of stemness characters, in vitro. Refining the in vitro culture can be particularly beneficial in the case of cord blood HSCs (CB-HSCs), as inadequate numbers in a single umbilical cord limits its therapeutic potential. In recent years, molecular dissection of HSC stemness has significantly improved in vitro hematopoietic stem and progenitor cells (HSPCs) culture. Despite such significant progress, lacunae exist in fully understanding all the underlying mechanisms and their interplay active in bona fide HSCs, and how it transforms when cells proliferate in culture. A new groundbreaking study titled “MYCT1 controls environmental sensing in human haematopoietic stem cells”, published in Nature in June 2024, sheds light on this complex field. Through a series of experiments, including knock-down, overexpression, single-cell RNA sequencing, and transplantation, the study identifies a previously unknown role of the MYC target 1 (MYCT1) protein in HSC maintenance. This protein acts as a crucial regulator of human HSCs, with high expression in primitive HSCs and subsequently downregulated during ex vivo culture. The study reveals that MYCT1 plays a vital role in moderating endocytosis and environmental sensing in HSCs, processes thereby essential for maintaining HSC stemness and function. This commentary will discuss the implications of the new findings for cord blood expansion in cell therapies and HSPC culture for gene therapy applications, providing valuable insights for the field of hematopoietic regenerative medicine.

Hemoglobin H (HbH) disease, a form of alpha‐thalassemia, poses significant clinical challenges due to its complex molecular underpinnings. It is characterized by reduced synthesis of the alpha‐globin chain. The integration of multi‐omics and precision medicine holds immense potential to comprehensively understand and capture interactions at the molecular and genetic levels. This review integrates current multi‐omics approaches and advanced technologies in HbH research. Furthermore, it delves into detailed pathophysiology and possible therapeutics in the upcoming future. We explore the role of genomics, transcriptomics, proteomics, and metabolomics studies, alongside bioinformatics tools and gene‐editing technologies like CRISPR/Cas9, to identify genetic modifiers, decipher molecular pathways, and discover therapeutic targets. Recent advancements are unveiling novel genetic and epigenetic modifiers impacting HbH disease severity, paving the way for personalized precision medicine interventions. The significance of multi‐omics research in unraveling the complexities of rare diseases like HbH is underscored, highlighting its potential to revolutionize clinical practice through precision medicine approaches. This paradigm shift can pave the way for a deeper understanding of HbH complexities and improved disease management. [ABSTRACT FROM AUTHOR]

Summary: Forests make immense contributions to societies in the form of ecological services and sustainable industrial products. However, they face major challenges to their viability and economic use due to climate change and growing biotic and economic threats, for which recombinant DNA (rDNA) technology can sometimes provide solutions. But the application of rDNA technologies to forest trees faces major social and biological obstacles that make its societal acceptance a 'wicked' problem without straightforward solutions. We discuss the nature of these problems, and the social and biological innovations that we consider essential for progress. As case studies of biological challenges, we focus on studies of modifications in wood chemistry and transformation efficiency. We call for major innovations in regulations, and the dissolution of method‐based market barriers, that together could lead to greater research investments, enable wide use of field studies, and facilitate the integration of rDNA‐modified trees into conventional breeding programs. Without near‐term adoption of such innovations, rDNA‐based solutions will be largely unavailable to help forests adapt to the growing stresses from climate change and the proliferation of forest pests, nor will they be available to provide economic and environmental benefits from expanded use of wood and related bioproducts as part of an expanding bioeconomy. [ABSTRACT FROM AUTHOR]

It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20–42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice. AAV mediated gene editing using CRISPR/Cas9 facilitates integration of a bypass coagulation factor VIIa and rescues bleeding phenotype in hemophilia B mice. Image created using Biorender.com. [Display omitted] • CRISPR/Cas9 mediated targeting of Rosa26 locus was found to have 20-42% efficiency in vitro. • Successful insertion of murine activated factor VII (mFVIIa) into the Rosa26 locus was achieved by CRISPR/Cas9 gene editing. • AAV mediated knock-in of bypass clotting factor mFVIIa using CRISPR/Cas9 mitigated bleeding phenotype in hemophilia B mice. • Increased FVIIa activity and shortened prothrombin time was attained after gene editing therapy in hemophilia B mice. [ABSTRACT FROM AUTHOR]

Banana is a premier fruit crop in many parts of the world especially Southeast Asia. The demand for banana has contributed to significant national income to primary banana producers in the SEA region such as the Philippines, Indonesia, Thailand, Vietnam, and Malaysia. However, the widely traded banana industry is plagued by numerous threats including pests and diseases, post-harvest issues and extreme climate vulnerability. To address these challenges, new breeding techniques such as gene editing have been explored for breeding programs to develop improved banana varieties. The first gene-edited non-browning banana has been deregulated in the Philippines recently, and more regulatory applications are expected to submit for approvals soon. Hence, it is timely to review the policy options for gene editing that have been adopted and discussed in the Southeast Asian countries and highlight the implications of differing regulatory approaches to gene editing for trading activities. Positive stakeholders' perceptions and public acceptance are key factors in allowing the benefits of gene editing and thus appropriate outreach strategies are important to gain acceptance and avoid the "GMO stigma" that may be associated with gene-edited products. [ABSTRACT FROM AUTHOR]

Abstract Gene‐editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene‐editing‐based therapy is unlikely to be successful; therefore, a multi‐gene knockout strategy is preferred for effective GBM inhibition. Here, a non‐invasive, biodegradable brain‐targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half‐life, efficient blood‐brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient‐derived GSC xenograft mouse models with negligible off‐target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi‐oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM.

CRISPR/Cas9, a potent genetic engineering tool widely adopted in agriculture, is capable of introducing new characteristics into plants on a large scale and without conventional breeding methods. Despite its remarkable efficiency, concerns have arisen regarding unintended consequences in uncontrolled environments. Our aim was to assess potential activity in organisms that could be exposed to genome editing in uncontrolled environments. We developed three scenarios, using irrigation, fumigation and fertilization as delivery methods, based on outdoor uses in agriculture, namely pest and disease control. Using publicly available software (Cas-OFFinder, NCBI Genome Data Viewer and STRING), off-target effects were predicted in multiple species commonly found in the agroecosystem, including humans (16 of 38 (42 %) sampled). Metabolic enrichment analysis (gene IDs), by connecting off-target genes into a physiological network, predicted effects on the development of nervous and respiratory systems. Our findings emphasize the importance of exercising caution when considering the use of this genome editing in uncontrolled environments. Unintended genomic alterations may occur in unintended organisms, underscoring the significance of understanding potential hazards and implementing safety measures to protect human health and the environment.

Immuno-oncology (IO) and targeted therapies, such as small molecule inhibitors, have changed the landscape of cancer treatment and prognosis; however, durable responses have been difficult to achieve due to tumor heterogeneity, development of drug resistance, and adverse effects that limit dosing and prolonged drug use. To improve upon the current medicinal armamentarium, there is an urgent need for new ways to understand, reverse, and treat carcinogenesis. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 is a powerful and efficient tool for genome editing that has shown significant promise for developing new therapeutics. While CRISPR/Cas9 has been successfully used for pre-clinical cancer research, its use in the clinical setting is still in an early stage of development. The purpose of this review is to describe the CRISPR technology and to provide an overview of its current applications and future potential as cancer therapies.

The current global food system, and in particular the livestock industry, has been effective at providing low-cost calories to large segments of the population, but it also causes significant harms and poses serious risks. In particular, the global food system currently likely causes billions of animals to suffer every year, significantly contributes to climate change, and threatens public health via the possibility of zoonotic disease. There are many other problems that have been identified with the livestock industry, but these three threats, which I refer to as the Big Three, are among the most urgent moral issues in the world. Significant progress could be made to address all three of these risks if the global population moved to a primarily plant-based diet. However, there are reasons to believe this possibility is unrealistic given current consumer preferences and political realities. As an alternative, one could ask whether an approach relying entirely on novel biotechnology could be used to address the urgent moral challenges of the global livestock industry without substantially changing the consumer experience or facing political backlash. In this paper I consider what such a scenario would look like, and argue that failing to address any one of these three major issues would be a serious moral failing. Though many other suggestions have been made looking at how biotechnology might address individual issues, this paper suggests that in order to avoid the need for difficult behavioral and political changes, biotechnological solutions would ultimately need to be developed that address welfare, environmental, and public health concerns. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The control of protein functions with light is valuable for spatiotemporal probing of biological systems. Current small‐molecule photo‐ modulation methods include the light‐induced uncaging of inhibitors and chromophore‐assisted light inactivation with reactive oxygen species (ROS). However, the constant target protein expression results in inadequate photo‐modulation efficiency, particularly for less potent inhibitors and chromophores. Herein, we report a novel bifunctional small‐molecule ligands strategy to photo‐modulate gene‐editing enzymes CRISPR/Cas9. A coumarin‐derived small‐molecule ligand Bhc‐BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity. Our results highlight the synergistic photo‐modulation with bifunctional small‐molecule ligands, which offers a valuable addition to current CRISPR/Cas9 photo‐modulation technologies and may extend to other protein classes. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The control of protein functions with light is valuable for spatiotemporal probing of biological systems. Current small‐molecule photo‐ modulation methods include the light‐induced uncaging of inhibitors and chromophore‐assisted light inactivation with reactive oxygen species (ROS). However, the constant target protein expression results in inadequate photo‐modulation efficiency, particularly for less potent inhibitors and chromophores. Herein, we report a novel bifunctional small‐molecule ligands strategy to photo‐modulate gene‐editing enzymes CRISPR/Cas9. A coumarin‐derived small‐molecule ligand Bhc‐BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity. Our results highlight the synergistic photo‐modulation with bifunctional small‐molecule ligands, which offers a valuable addition to current CRISPR/Cas9 photo‐modulation technologies and may extend to other protein classes. [ABSTRACT FROM AUTHOR]

He Jiankui announced to the world in November 2018 that his team had genetically edited twin human embryos that were then brought to term. Recruiting participants through an HIV outreach group and using CRISPR/Cas9 technology, He targeted the CCR5 genes claiming this would make the children immune to HIV. One way to understand He's case is through Charis Thompson's concept of ethical choreography, which shows how scientists 'invent around' potential ethical objections to their work. In particular, such a focus on ethical choreography traces how individual scientists can exploit ambiguity in institutional boundaries to recombine different logics to advance their vision of good, innovative, and ethical science. He's actions can be seen to been the result of his traversal of blurred boundaries that demarcate science/market and science/medicine in order to recombine academic, market, medical, and cultural logics. This combination of logics is seen in ethical justifications that He put forth for his experiment, which provoked much criticism and controversy, but should nonetheless be taken seriously and placed in context. While He's vision of good science was rejected, examples of ethical choreography can be found in other instances of biomedical innovation and there remains the potential for other scientists to pick up where He left off. Following the ethical choreography of scientists also allows for more specificity in discussions about what and when boundaries should be strengthened or relaxed in order to advance a more equitable vision of science and technology. [ABSTRACT FROM AUTHOR]

This dataset contains the gene sequences of the small and large sub-unit of the hydrogenase enzyme obtained from the annotated genome of Rhodobacter johrii MAY2. The whole genome sequence of the isolate was performed using SEED genome viewer on the Rapid Annotation using the Subsystem Technology (RAST) platform. Concurrently, guide RNA sequences and primers were meticulously crafted using the CHOPCHOP v.3.0 web tool, specifically designed for the precise editing and amplification of the target genes. The primers were optimized via gradient PCR to determine appropriate amplification conditions. Furthermore, the guide RNA was tested via in-vitro cleavage assay, gauging its efficacy in cleaving the intended target genes. The dataset, including the optimization and the cleavage assay, was deposited in Mendeley Data with DOI no: 10.17632/rcx3mcssnx.2.

Abstract Background Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Results Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. Conclusions Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

The current global food system, and in particular the livestock industry, has been effective at providing low-cost calories to large segments of the population, but it also causes significant harms and poses serious risks. In particular, the global food system currently likely causes billions of animals to suffer every year, significantly contributes to climate change, and threatens public health via the possibility of zoonotic disease. There are many other problems that have been identified with the livestock industry, but these three threats, which I refer to as the Big Three, are among the most urgent moral issues in the world. Significant progress could be made to address all three of these risks if the global population moved to a primarily plant-based diet. However, there are reasons to believe this possibility is unrealistic given current consumer preferences and political realities. As an alternative, one could ask whether an approach relying entirely on novel biotechnology could be used to address the urgent moral challenges of the global livestock industry without substantially changing the consumer experience or facing political backlash. In this paper I consider what such a scenario would look like, and argue that failing to address any one of these three major issues would be a serious moral failing. Though many other suggestions have been made looking at how biotechnology might address individual issues, this paper suggests that in order to avoid the need for difficult behavioral and political changes, biotechnological solutions would ultimately need to be developed that address welfare, environmental, and public health concerns.

Background: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Results: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. Conclusions: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Automated techniques for mammalian cell engineering are needed to examine a wide range of unique genetic perturbations especially when working with precious patient samples. An automated and miniaturized technique making use of digital microfluidics to electroporate a minimal number of mammalian cells (≈40 000) at a time on a scalable platform is introduced. This system functions by merging three droplets into a continuous droplet chain, which is called a triDrop. In the triDrop configuration, the outer droplets are comprised of high‐conductive liquid while an inner or middle droplet comprising of low‐conductivity liquid that contains the cells and biological payloads. In this work, it is shown that applying a voltage to the outer droplets generates an effective electric field throughout the tri‐droplet structure allowing for insertion of the biological payload into the cells without sacrificing long‐term cell health. This technique is shown for a range of biological payloads including plasmids, mRNA, and fully formed proteins being inserted into adherent and suspension cells which include primary T‐cells. The unique features of flexibility and versatility of triDrop show that the platform can be used for the automation of multiplexed gene edits with the benefits of low reagent consumption and minimal cell numbers. [ABSTRACT FROM AUTHOR]

The soybean (Glycine max L.) is an important crop with high agronomic value. The improvement of agronomic traits through gene editing techniques has broad application prospects in soybean. The polyethylene glycol (PEG)-mediated cell transfection has been successfully used to deliver the CRISPR/Cas9-based ribonucleoprotein (RNP) into soybean protoplasts. However, several downstream analyses or further cell regeneration protocols might be hampered by PEG contamination within the samples. Here in this study, we attempted to transfect CRISPR/Cas9 RNPs into trifoliate leaf-derived soybean protoplasts using Neon electroporation to overcome the need for PEG transfection for the first time. We investigated different electroporation parameters including pulsing voltage (V), strength and duration of pulses regarding protoplast morphology, viability, and delivery of CRISPR/Cas9. Electroporation at various pulsing voltages with 3 pulses and 10 ms per pulse was found optimal for protoplast electro-transfection. Following electro-transfection at various pulsing voltages (500 V, 700 V, 1,000 V, and 1,300 V), intact protoplasts were observed at all treatments. However, the relative frequency of cell viability and initial cell divisions decreased with increasing voltages. Confocal laser scanning microscopy (CLSM) confirmed that the green fluorescent protein (GFP)-tagged Cas9 was successfully internalized into the protoplasts. Targeted deep sequencing results revealed that on-target insertion/deletion (InDel) frequencies were increased with increasing voltages in protoplasts electro-transfected with CRISPR/Cas9 RNPs targeting constitutive pathogen response 5 (CPR5). InDel patterns ranged from +1 bp to -6 bp at three different target sites in CPR5 locus with frequencies ranging from 3.8% to 8.1% following electro-transfection at 1,300 V and 2.1% to 3.8% for 700 V and 1,000 V, respectively. Taken together, our results demonstrate that the CRISPR/Cas9 RNP system can be delivered into soybean protoplasts by the Neon electroporation system for efficient and effective gene editing. The electro-transfection system developed in this study would also further facilitate and serve as an alternative deliverymethod for DNA-free genome editing of soybean and other related species for genetic screens and potential trait improvement. [ABSTRACT FROM AUTHOR]

Introduction: Although pulses are nutritious foods containing high amounts of protein, fiber and phytochemicals, their consumption and use in the food industry have been limited due to the formation of unappealing flavors/aromas described as beany, green, and grassy. Lipoxygenase (LOX) enzymes are prevalent among pulse seeds, and their activity can lead to the formation of specific volatile organic compounds (VOCs) from certain polyunsaturated fatty acids (PUFAs). As a widespread issue in legumes, including soybean, these VOCs have been linked to certain unappealing taste perception of foods containing processed pulse seeds. Methods: To address this problem in pea and as proof of principle to promote the wider use of pulses, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) construct was designed to create null alleles (knockouts) of PsLOX2 which had been implicated in the generation of VOCs in peas. Results and discussion: Successful CRISPR/Cas9-mediated LOX gene editing of stable transgenic pea lines (TGP) was confirmed by DNA sequencing of the wild type (WT) and TGP pslox2 mutant lines. These lines were also assessed for LOX activity, PUFA levels, and VOCs. Compared to WT peas, the TGP lines showed a significant reduction (p < 0.05) in LOX activity and in the concentration of key VOCs, including hexanal, 2-hexenal, heptanal, (E)-2-heptenal, (E,E)-2,4-heptadienal, 1-octen-3-ol, octanal, (E)-2-octenal (E,E)-2,4-nonadienal and furan-2-pentyl. The content of two essential PUFAs, linoleic and α-linolenic acids, the known substrates of LOX in plants, was higher in TGP flours, indicating the efficacy of the CRISPR-mediated gene editing in minimizing their oxidation and the further modification of PUFAs and their products. The collection of VOCs from the headspace of ground pea seeds, using a portable eNose also distinguished the TGP and WT lines. Multiple regression analysis showed that LOX activity correlated with the two VOCs, heptanal and (E,E)-2,4-heptadienal in pea flours. Partial Least Squares Regression (PLS-R) plot for selected PUFAs, VOCs, and sensor responses in WT and TGP lines showed distinct clusters for WT and TGP lines. Together this data demonstrates the utility of CRISPR mediated mutagenesis of PsLOX2 to quickly improve aroma and fatty acid (FA) profiles of pea seeds of an elite Canadian variety. [ABSTRACT FROM AUTHOR]

Millions of hectares of cover crops are planted in the U.S. and European Union to manage soil erosion, soil fertility, water quality, weeds, and climate change. Although only a small percentage of cover crops are harvested, the growing cover crop planting area provides a new biomass source to the biofuel industry to produce bioenergy. Oilseed crops such as rapeseed, sunflower, and soybean are commodities and have been used to produce biodiesel and sustainable aviation fuel (SAF). Other cover crops such as cereal rye, clover, and alfalfa, have been tested on small or pilot scales to produce cellulosic ethanol, biogas, syngas, bio-oil, and SAF. Given the various biofuel products and pathways, this review aimed to provide a comprehensive comparison of biofuel yield from different cover crops and an overview of the technologies that have been employed to improve biofuel yield. It was envisioned that gene-editing tools might be revolutionary to the biofuel industry, the work on cover crop supply chain will be critical for system scaleup, and high-tolerant technologies likely will be needed to handle the high compositional heterogeneity and variability of cover crop biomass for biofuel. [ABSTRACT FROM AUTHOR]

New plant breeding techniques may play an important role in improving food quality, global food security and sustainability. Previous breeding techniques have, however, met with substantial resistance from society. This study examined the role of associations and deliberation in the evaluation of breeding techniques. Breeding techniques studied included conventional breeding, gene-editing, genetic modification (cisgenesis and transgenesis), marker-assisted breeding and synthetic biology. By using focus group discussions that included individual tasks, we found that when participants relied on their spontaneous associations, gene-editing was evaluated similarly as genetic modification. However, after information provision and group discussion, gene-editing was preferred over genetic modification. Perceived naturalness was found to be the main reason for obtaining different levels of acceptance, not only between gene-editing and genetic modification but across all breeding techniques examined. These findings highlight the importance of associations and show that beliefs about naturalness remain crucial in understanding how consumers evaluate breeding techniques. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes. [ABSTRACT FROM AUTHOR]

In December 2019, a Chinese court sentenced Jiankiu He, the scientist who used genome editing to create three babies via in vitro fertilization (IVF), to three years in prison for practicing medicine without a license. The court claimed that He violated Chinese law when he forged ethical review documents and misled doctors into unknowingly implanting genetically altered embryos into two women the previous year. He stated that he had taken a leave from his university prior to starting the clinical stage of his project, and that he financed that part of the study from his own personal savings, therefore seemingly absolving his university and Chinese authorities of any responsibility in the affair. But to many observers, it seems inconceivable that Jiankui He could have engaged in this research without at least tacit government approval. This paper explores the sources of funding for He's experiment, in an attempt to clarify what Chinese authorities knew or should have known – and whether it is conceivable that He could have completed the clinical phase of the project with no direct government knowledge. It then analyzes the roles that China's science policy and scientific culture played in enabling He to act as he did. [ABSTRACT FROM AUTHOR]

The soybean (Glycine max L.) is an important crop with high agronomic value. The improvement of agronomic traits through gene editing techniques has broad application prospects in soybean. The polyethylene glycol (PEG)-mediated cell transfection has been successfully used to deliver the CRISPR/Cas9-based ribonucleoprotein (RNP) into soybean protoplasts. However, several downstream analyses or further cell regeneration protocols might be hampered by PEG contamination within the samples. Here in this study, we attempted to transfect CRISPR/Cas9 RNPs into trifoliate leaf-derived soybean protoplasts using Neon electroporation to overcome the need for PEG transfection for the first time. We investigated different electroporation parameters including pulsing voltage (V), strength and duration of pulses regarding protoplast morphology, viability, and delivery of CRISPR/Cas9. Electroporation at various pulsing voltages with 3 pulses and 10 ms per pulse was found optimal for protoplast electro-transfection. Following electro-transfection at various pulsing voltages (500 V, 700 V, 1,000 V, and 1,300 V), intact protoplasts were observed at all treatments. However, the relative frequency of cell viability and initial cell divisions decreased with increasing voltages. Confocal laser scanning microscopy (CLSM) confirmed that the green fluorescent protein (GFP)-tagged Cas9 was successfully internalized into the protoplasts. Targeted deep sequencing results revealed that on-target insertion/deletion (InDel) frequencies were increased with increasing voltages in protoplasts electro-transfected with CRISPR/Cas9 RNPs targeting constitutive pathogen response 5 (CPR5). InDel patterns ranged from +1 bp to −6 bp at three different target sites in CPR5 locus with frequencies ranging from 3.8% to 8.1% following electro-transfection at 1,300 V and 2.1% to 3.8% for 700 V and 1,000 V, respectively. Taken together, our results demonstrate that the CRISPR/Cas9 RNP system can be delivered into soybean protoplasts by the Neon electroporation system for efficient and effective gene editing. The electro-transfection system developed in this study would also further facilitate and serve as an alternative delivery method for DNA-free genome editing of soybean and other related species for genetic screens and potential trait improvement.

IntroductionAlthough pulses are nutritious foods containing high amounts of protein, fiber and phytochemicals, their consumption and use in the food industry have been limited due to the formation of unappealing flavors/aromas described as beany, green, and grassy. Lipoxygenase (LOX) enzymes are prevalent among pulse seeds, and their activity can lead to the formation of specific volatile organic compounds (VOCs) from certain polyunsaturated fatty acids (PUFAs). As a widespread issue in legumes, including soybean, these VOCs have been linked to certain unappealing taste perception of foods containing processed pulse seeds.MethodsTo address this problem in pea and as proof of principle to promote the wider use of pulses, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) construct was designed to create null alleles (knockouts) of PsLOX2 which had been implicated in the generation of VOCs in peas.Results and discussionSuccessful CRISPR/Cas9-mediated LOX gene editing of stable transgenic pea lines (TGP) was confirmed by DNA sequencing of the wild type (WT) and TGP pslox2 mutant lines. These lines were also assessed for LOX activity, PUFA levels, and VOCs. Compared to WT peas, the TGP lines showed a significant reduction (p < 0.05) in LOX activity and in the concentration of key VOCs, including hexanal, 2-hexenal, heptanal, (E)-2-heptenal, (E,E)-2,4-heptadienal, 1-octen-3-ol, octanal, (E)-2-octenal (E,E)-2,4-nonadienal and furan-2-pentyl. The content of two essential PUFAs, linoleic and α-linolenic acids, the known substrates of LOX in plants, was higher in TGP flours, indicating the efficacy of the CRISPR-mediated gene editing in minimizing their oxidation and the further modification of PUFAs and their products. The collection of VOCs from the headspace of ground pea seeds, using a portable eNose also distinguished the TGP and WT lines. Multiple regression analysis showed that LOX activity correlated with the two VOCs, heptanal and (E,E)-2,4-heptadienal in pea flours. Partial Least Squares Regression (PLS-R) plot for selected PUFAs, VOCs, and sensor responses in WT and TGP lines showed distinct clusters for WT and TGP lines. Together this data demonstrates the utility of CRISPR mediated mutagenesis of PsLOX2 to quickly improve aroma and fatty acid (FA) profiles of pea seeds of an elite Canadian variety.

Photo by ANIRUDH on Unsplash INTRODUCTION Genes are the most foundational physical unit of a human, coding for every phenotypic characteristic that a human exhibits.[1] Thus, some view the editing of genes as the alteration of one’s physical nature. The extent to which gene-editing, using technology such as CRISPR, alters one's identity is an open issue. Answering this question requires exploring what constitutes human identity (physical or mental forms, or both) and the role DNA plays. This philosophical question concerns the role of the body in the conception of oneself and how fundamental changes to one’s body alter that conception. As gene-editing technologies continue to be refined and implemented in humans, this question is especially relevant. Its answer can alter the decisions society makes about the use of gene-editing technology in humans. Alterations to one’s physical form through gene-editing change one’s physical makeup. Gene-editing causes modifications that span both realms of one’s sense of self because physical identity can affect mental identity. This paper explores philosophical approaches to identity and the relationship between body and mind, arguing that physical changes have significant capacity to alter emotions and mental status in ways that may shape identity. I. CRISPR and Gene-Editing Capability Scientists can use gene-editing to rectify the genetic flaws that cause genetic disorders. Trisomy 21, or Down Syndrome, for example, is caused by an additional 21st chromosome in a person’s genome. Deletion of this extra chromosome through gene-editing would erase the disease, changing the person’s genomic foundation and life. They would no longer need to manage the effects of trisomy 21. Gene-editing in the case of trisomy 21 impacts identity. An analysis of perspectives on the link between trisomy gene-editing and identity revealed a split between those who viewed trisomy as separate from identity and those who viewed it as fundamental. Participants favoring a connection between trisomy and identity were more likely to oppose gene-editing, as they believed it would bring fundamental changes to who the person is.[2] Discussion of the extent to which gene-editing impacts identity is important in informing these opinions and, in turn, the policies society implements on the use of gene-editing. Somatic gene-editing, performed in existing people rather than an embryo, proposes a unique case. Many view somatic editing as “similar to other medical treatments” rather than a complete alteration to one’s identity because it is only used in cells in specific tissues to fix symptoms, not the entire disease. For trisomy 21, somatic gene-editing will only be possible in specific tissues and cells of the body, such as in the nerve cells that cause muscle weakness in Down Syndrome patients, since it is not yet possible to change every cell carrying the genetic flaw.[3] Researchers can treat other diseases more completely with gene-editing. To fix the single nucleotide mutation that causes sickle cell disease, for example, researchers can edit the red bone marrow cells extracted from patients, edit them using CRISPR, and reintroduce them into the patient.[4] Gene-editing enables a more comprehensive solution for sickle cell disease in existing people, but it entails the same genome editing as with trisomy. Regardless of the disease or somatic or germline cell targeting, gene-editing alters the DNA a human carries. By exploring the most basic implications of that alteration on human identity, this paper will define the relationship between gene-editing and identity. II. Views on Physical Identity Some may view a human’s physical nature as their only nature and thus the sole component of identity. Reductive physicalism proposes that the world is made of only physical components.[5] Reductive physicalists challenge the existence of the mind or a higher power by arguing that all processes can be broken down into their physical components. By this reasoning, humans are a collection of atoms and molecules compartmentalized into cells that perform all human functions. By suggesting that humans are entirely physical in nature, this theory proposes that one’s physicality must constitute identity. In A Treatise of Human Nature, David Hume alludes to the essential role of physicality in identity. Although Hume also discusses the role of a non-physical mind in the processing of perceptions, Hume states that “after the dissolution of my body, I should be entirely annihilated.”[6] The destruction of one’s being, one’s identity, when one ceases to physically exist reinforces the idea that humans are solely physical in nature. Applying reductive physicalism, the dependence of identity on physicality supports that changing one’s physicality through gene-editing will change one’s identity. In Being and Nothingness, Jean-Paul Sartre argues that a human confirms his existence when he sees “the other” looking at him, becoming an object rather than a subject. Describing one’s sudden self-consciousness and resulting shame, Sartre poses “the look” as essential in one’s formation of a sense of self.[7] Since the body is recognized by “the other,” the pre-reflective cogito suggests that, without the body, one would not be able to recognize their own existence. Under Sartre’s argument, a physical body is a precursor to identity. However, gene-editing by altering the physical characteristics of a human, such as the shape of red blood cells in sickle cell disease patients, generally leaves the body intact. One would still be perceived by “the other” after gene-editing and have their identity confirmed. Sartre’s “the look” reinforces the importance of the body. Yet gene-editing would not impact one’s being an object of another. III. Mind and Body Combine in Human Identity In philosophy, mind-body dualism means that humans are composed of mental and physical substances. These forms of an individual are distinct yet inextricably interconnected.[8] René Descartes introduced mind-body dualism to argue the existence of a mind without disputing the existence of a body. In his Cogito Theory, Descartes states, “I think, therefore I am.” According to Descartes, the mind is an indivisible substance that coexists with but is distinct from the body because it lacks shape and physicality.[9] Descartes confirms his existence by the fact that his mind is a “thinking thing” rather than by the other’s recognition of one’s body, as Sartre does. The idea of a non-physical aspect to humans challenges reductive physicalism and provides the space for something more than the body to define a human. The mind confirms existence and thereby grants identity, suggesting not only that the mind exists, but also that it is more central to identity than the body. This view aligns with current views about identity. Since Descartes regards the mind and body as two distinct substances, in his view, the physical changes caused by gene-editing do not directly influence the mind. Gene-editing alters the molecules in genes, but according to dualism, the mind is not composed of atoms like the body is. A genetic change would not penetrate the mind. The distinction between the mental and physical aspects of identity demonstrates how gene-editing may not have substantial power over one’s identity. Since Descartes prioritizes the mental aspect of identity over the physical, gene-editing’s effect on identity appears even more limited. IV. Mind-Body Philosophies Other philosophical viewpoints further support a mental aspect of human identity. In De Anima, Aristotle delineates that the body is a manifestation of the mind, which is a manifestation of the soul.[10] Although the mind and body are two essential, coexistent aspects of a human, the body comes from, and thus relies on, the mind. The mind, as the origin of the body, frames it as more central to human existence. This postulates that the changes to physical identity induced by gene-editing technologies may not directly influence the mental aspect of identity because the mind does not stem from, and thus is not dependent on, the body. The mind’s more direct connection to the soul cements its larger role in identity because non-secular philosophy views the soul as the core, eternal aspect of a human. The superiority of the mind over the body regarding identity demonstrates gene-editing’s limited influence on identity. In A Treatise of Human Nature, Hume suggests that identity is formed based on perceptions, reinforcing the role of the mind. Perceptions, which originate as a physical sensation, inspire feelings and thoughts, an aspect of one’s mind. Hume gives the example of feeling the warmth of a fire.[11] The emotions that arise are processed and stored in the mind. The accumulation of perceptions from different experiences shapes one’s identity. A traumatic event or significant accomplishment often becomes part of one’s sense of self because of the perceptions one acquires. Hume’s proposition that successive perceptions forms the mind, which are important to identity, further demonstrates that the mind is an aspect of identity. Under such a viewpoint, gene-editing would appear restricted in its ability to change identity, as it alters the body but not the mind. DNA cannot change the experiences and thoughts encoded in one’s mind. One may argue that scars on one’s body, rather than thoughts in one’s mind, are the marks of one’s perceptions, like a burn mark from touching a hot stove. However, the memories and emotions of an experience that a scar represents are deeper than the scar itself. A scar on a person has more meaning than a scar on a wax figure, a purely physical being, because of the mental associations with the scar in the person’s mind. Thoughts residing in the mind contribute to one’s identity, highlighting the role of the mind in a human's sense of self. Although gene-editing in non-neuronal cells cannot directly alter one’s thoughts, the processing of perceptions to form mental identity depends on the physical aspect of humans. Hume proposes that the body mediates the outside world and the mind, detecting stimuli and relaying information to the mind. This communicative relationship between the mind and the body, expressed in mind-body dualism, proposes that physical changes caused by gene-editing could affect how and when external stimuli are detected. This indirect consequence that gene-editing can have on the mind opens the discussion of how physical genetic changes could affect mental identity despite the immaterial nature of the mind. V. Current Scientific Understanding of the Mind-Body Relationship There is significant medical research on the relationship between the physical body and emotions like happiness, anger, and depression. Modern science sees many emotions as linked to the physical. Although gene-editing does not directly change the mind, it can be affected by those physical changes. It is well-known that there is a “functionally integrated relationship between mind and body.”[12] This “relationship” suggests that the mind and body do not function separately but rather influence each other. For example, replacing bad gut bacteria with good gut bacteria in animals and humans can “significantly alter mood and emotional functions.”[13] Gut bacteria is a purely physical component of the body, while mood and emotion are more mental. The influence of the physical on the mental illustrates the effect of the body’s state on the mind. The two-way communication that the mind and body exhibit have important consequences for the role of DNA in identity. Genetic alterations caused by CRISPR, for example, may affect the mental aspect of identity. Researchers can use CRISPR to cure a sickle cell disease patient by changing just one nucleotide in a human’s genome.[14] The editing of the patient’s genome changes their physical identity because genes are a physical component of humans, but mental changes can also result. No longer suffering from the debilitating pain of a chronic disease, the patient’s mood and outlook on life may shift. The integrated mind-body relationship suggests that gene-editing may have a broader impact on identity than anticipated, affecting both the body and, indirectly, the mind. VI. Mind and Brain Philosophy The effects of gene-editing on the mind can be more direct when the genes edited are associated with the brain. One theory explaining this phenomenon is the Mind-Brain Identity Theory. This theory postulates that “states and processes of the mind are identical to states and processes of the brain” even though the mind is non-physical.[15] Therefore, changes to the brain will correlate with changes to the mind and, in turn, affect mental identity. Using CRISPR to correct the genes that cause Huntington's Disease, for example, would improve the physical conditions in the brain that cause its symptoms. According to the Mind-Brain Identity theory, it would improve one’s mental state as a result. If a mistake occurs and the condition worsens, or researchers edit beyond correcting the disorder, the negative consequences in the brain would correlate with a worsened state of mind. The direct correlation between the mind and brain increases the risks of gene-editing. The Mind-Brain Theory amplifies gene-editing’s effect on identity because it suggests physical changes can have not only indirectly, but also directly, impact on mental identity. CONCLUSION Gene-editing alters the physical body. Due to the integrated mind-body relationship, it can also directly and indirectly modify mental identity. Mind-body dualism, as opposed to reductive physicalism, yields an understanding of identity that parallels the way mind-body connections are currently understood. Current science suggests that mood and emotion may be more squarely tied to the physical. Identity likely depends on some combination of the physical and mental. Framing mental aspects, including emotions, personality, and thoughts, as immaterial and genes as physical could lead to the overuse of gene-editing technology for physical purposes like disease prevention, solutions, or enhancement. Mind-body dualism forms the foundation for gene-editing’s nuanced, far-reaching impact on one’s sense of self. Identity defines an individual, both to themselves and others. The potential for unknown or undesired effects of gene-editing on identity calls for balancing the technology’s benefit to human health with its potentially negative impact on identity. The philosophical and ethical underpinnings should help inform public policy and scientific engagement as gene-editing becomes more influential in medicine. - [1] “The predominant current day meaning of genotype is some relevant part of the DNA passed to the organism by its parents. The phenotype is the physical and behavioral traits of the organism, for example, size and shape, metabolic activities, and patterns of movement.” Taylor, Peter and Richard Lewontin, "The Genotype/Phenotype Distinction", The Stanford Encyclopedia of Philosophy (Summer 2021 Edition), Edward N. Zalta (ed.), URL = . [2] Elliott, Kathryn, et al. “‘I Wouldn’t Want Anything That Would Change Who He Is.’ the Relationship between Perceptions of Identity and Attitudes towards Hypothetical Gene-Editing in Parents of Children with Autosomal Aneuploidies.” SSM - Qualitative Research in Health, Elsevier, Dec. 2022, www.sciencedirect.com/science/article/pii/S2667321522001135. [3] Watson-Scales, Sheona et al. “Analysis of motor dysfunction in Down Syndrome reveals motor neuron degeneration.” PLoS genetics vol. 14,5 e1007383. 10 May. 2018, doi:10.1371/journal.pgen.1007383 [4] Cross, Ryan. “CRISPR edits sickle cell mutation” C&EN Global Enterprise 94 (41), 5-5, October 17, 2016. https://pubs.acs.org/doi/10.1021/cen-09441-notw1 [5] Stoljar, Daniel. “Physicalism.” Stanford Encyclopedia of Philosophy. Stanford University, May 25, 2021. https://plato.stanford.edu/entries/physicalism/. [6] Hume, David. “Of Personal Identity.” Essay. In A Treatise of Human Nature. Project Gutenberg, n.d. [7] Sartre, Jean-Paul. Being and Nothingness, n.d. [8] Descartes, René. Meditations on First Philosophy. Yale University, n.d. [9] Descartes, René. Meditations on First Philosophy. Yale University, n.d. [10] Aristotle. “The Internet Classics Archive: De Anima (On the Soul) by Aristotle.” The Internet Classics Archive | On the Soul by Aristotle, n.d. http://classics.mit.edu/Aristotle/soul.html. [11] Hume, David. “Of Personal Identity.” Essay. In A Treatise of Human Nature. Project Gutenberg, n.d. [12] News. “A Neurosurgeon and a Philosopher Debate Mind vs. Body.” Mind Matters, February 28, 2022. https://mindmatters.ai/2022/02/a-neurosurgeon-and-a-philosopher-debate-mind-vs-body/. [13] Jasanoff, Alan. The Biological Mind How Brain, Body, and Environment Collaborate to Make Us Who We Are. Cambridge: Basic Books, 2018. [14] Frangoul, Haydar et al. “CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.” The New England journal of medicine vol. 384,3 (2021): 252-260. doi:10.1056/NEJMoa2031054 [15] Smart, J. J. C. “The Mind/Brain Identity Theory.” Stanford Encyclopedia of Philosophy. Stanford University, May 18, 2007. https://plato.stanford.edu/entries/mind-identity/.

SUMMARY: Precise gene‐editing using CRISPR/Cas9 technology remains a long‐standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi‐type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low‐expression genes (CrTET1 and CrKU80), the introduction of a FLAG‐HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live‐cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3′‐UTR of MAA7 and VIPP1 resulted in a stable knock‐down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications. [ABSTRACT FROM AUTHOR]

High-density lipoprotein (HDL) is an enigmatic member of the plasma lipid and lipoprotein transport system, best known for its ability to promote the reverse cholesterol efflux and the unloading of excess cholesterol from peripheral tissues. More recently, data in experimental mice and humans suggest that HDL may play important novel roles in other physiological processes associated with various metabolic disorders. Important parameters in the HDL functions are its apolipoprotein and lipid content, further reinforcing the principle that HDL structure defines its functionality. Thus, based on current evidence, low levels of HDL-cholesterol (HDL-C) or dysfunctional HDL particles contribute to the development of metabolic diseases such as morbid obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. Interestingly, low levels of HDL-C and dysfunctional HDL particles are observed in patients with multiple myeloma and other types of cancer. Therefore, adjusting HDL-C levels within the optimal range and improving HDL particle functionality is expected to benefit such pathological conditions. The failure of previous clinical trials testing various HDL-C-raising pharmaceuticals does not preclude a significant role for HDL in the treatment of atherosclerosis and related metabolic disorders. Those trials were designed on the principle of "the more the better", ignoring the U-shape relationship between HDL-C levels and morbidity and mortality. Thus, many of these pharmaceuticals should be retested in appropriately designed clinical trials. Novel gene-editing-based pharmaceuticals aiming at altering the apolipoprotein composition of HDL are expected to revolutionize the treatment strategies, improving the functionality of dysfunctional HDL. [ABSTRACT FROM AUTHOR]

Genetic modification enables modification of target genes or genome structure in livestock and experimental animals. These technologies have not only advanced bioscience but also improved agricultural productivity. To introduce a foreign transgene, the piggyBac transposon element/transposase system could be used for production of transgenic animals and specific target protein-expressing animal cells. In addition, the clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9 (CRISPR-Cas9) system have been utilized to generate chickens with knockout of G0/G1 switch gene 2 (G0S2) and myostatin, which are related to lipid deposition and muscle growth, respectively. These experimental chickens could be the invaluable genetic resources to investigate the regulatory pathways and mechanisms of improvement of economic traits such as fat quantity and growth. The gene-edited animals could also be applicable to the livestock industry.

Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animal agriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells, somatic cell nuclear transfer, cryopreservation, and nonsurgical embryo transfer. The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.

Transthyretin(TTR) protein is a tetramer protein, synthesized mainly by the liver. TTR can be misfolded and deposited as amyloid fibrilae and deposited in the myocardial interstroma leading to transthyroxin amyloidosis cardiomyopathy (ATTR-CM). ATTR-CM was included in China's First List of Rare Diseases. Therapeutic strategies for ATTR-CM include blocking TTR synthesis in the liver, stabilizing TTR tetramers and destroying TTR fibra. Small molecule drugs such as tafamidis and diflunisal offer new treatment options for patients. Chlorobenzolic acid became the first drug approved by the U.S. Food and Drug Administration for the treatment of ATTR-CM. Small interfering RNA(siRNA)patisiran and antisense oligonucleotide (ASO)inotersen block TTR expression in the liver and have been approved for the treatment of ATTR variant polyneuropathy (ATTRv-PN)and are in phase Ⅲ trials for the treatment of ATTR-CM. Other siRNA drugs, vutrisiran, and ASO, eplontersen, are being evaluated for clinical efficacy. This article reviews the development of RNA-targeted therapeutics and gene-editing drugs using CRISPR-Cas9.

The Chinese government has issued a series of new policies to make it easier to industrialize gene-edited crops. However, whether technological advantages will eventually translate into industrial advantages and whether farmers will soon have access to gene-edited varieties partly depends on seed companies’ willingness to produce and sell gene-edited varieties to farmers and to invest in developing their own gene-edited varieties. This study utilizes data from a survey of 111 seed companies collected in 2019 before the implementation of new regulations. This study provides empirical evidence on whether gene-edited crops will be available to farmers. The results show that the number of companies conducting research on gene-edited crops is limited, mostly to large companies. Approximately 55% of seed company managers would consider developing and selling gene-edited crops modified by SDN-1 and SDN-2 site-directed nuclease genome editing without external genetic material, whereas 46% support crops modified by SDN-3, which require gene replacement or foreign deoxyribonucleic acid (DNA) insertion and are regulated as genetically modified organisms (GMOs). The regression results show that large companies and companies with well-educated researchers are more likely to support and develop gene-editing technology. Past GM investment experience and collaboration with public institutions in gene-editing research increases the probability of company investment in gene editing R&D. These results suggest that gene-edited cultivars are more likely to be produced and sold to farmers in the future than GMOs, and that gene-edited agricultural products could have a significant market share of the seed market in the future.

The genome is the blueprint for life, and over the past decade, CRISPR has become a very powerful method for editing our genetic makeup. In this article, we will explore the importance of CRISPR in developing breakthrough therapies for monogenic conditions and neurodegenerative diseases, and for enhancing the effectiveness of immuno-oncology.

Objective: Concerns about off-target effects (OTEs) of genomic DNA cleavages by gene-editing enzymes have been raised, especially for OTEs that go undetected due to technical limitations. Since no explicit guidelines have been in place for risk assessment of OTEs, the regulatory authorities' concept of an acceptable evaluation scheme for OTEs in the investigational drug application (IND) has not been clear. Here, we clarify the regulatory expectations by examining reports on OTE evaluations of leading gene-editing products that have achieved IND clearance. Methods: We collected and analyzed the gene-editing products that have entered clinical trials by searching on ClinicalTrials.gov and EU-Clinical-Trial-Registries, and related reports for OTE evaluations from Google Scholar, PubMed, and the developers' websites. Results: We found a common two-step verification method used for different products at the preclinical stage. First, numerous potential off-target loci (POLs) are listed with state-of-the-art high-sensitivity detection methods and theoretical screens; Second, these OTEs are checked by amplicon sequencing of the POLs after treatment by enzymes in in vitro models close to clinical use conditions. Only the OTEs that can be detected and verified are addressed in the risk assessment in the translational phase from preclinical to clinical study. Discussion: Here, we describe a clear scheme for risk assessment of OTEs at the key translational phase, based on the common features in protocols for gene-editing products that were cleared for use in clinical trials. This report will provide a guide for those newly attempting to conduct clinical development in this field. [ABSTRACT FROM AUTHOR]

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1DEL or elF4E_3DEL) and double (elF4E_1-3DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3DEL and elF4E_1DEL mutants. However, homozygous elF4E_3DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production. [ABSTRACT FROM AUTHOR]

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Agriculture has supported human life from the beginning of civilization, despite a plethora of biotic (pests, pathogens) and abiotic (drought, cold) stressors being exerted on the global food demand. In the past 50 years, the enhanced understanding of cellular and molecular mechanisms in plants has led to novel innovations in biotechnology, resulting in the introduction of desired genes/traits through plant genetic engineering. Targeted genome editing technologies such as Zinc-Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as powerful tools for crop improvement. This new CRISPR technology is proving to be an efficient and straightforward process with low cost. It possesses applicability across most plant species, targets multiple genes, and is being used to engineer plant metabolic pathways to create resistance to pathogens and abiotic stressors. These novel genome editing (GE) technologies are poised to meet the UN’s sustainable development goals of “zero hunger” and “good human health and wellbeing.” These technologies could be more efficient in developing transgenic crops and aid in speeding up the regulatory approvals and risk assessments conducted by the US Departments of Agriculture (USDA), Food and Drug Administration (FDA), and Environmental Protection Agency (EPA).

The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.