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Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr−/−) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr−/− males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr−/− males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.

The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 gene in induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations on plasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.

The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.

Climate change has been projected to negatively affect genetic resources of the forests worldwide. Thus, integrating biotechnological innovations with traditional forest conservation strategies offers as a promising pathway to enhance the resilience of forest genetic resources. Biodiversity informatics plays a crucial role in managing and analysing data, supporting informed decision-making in forest management and conservation. Advancements in genomics and sequencing technologies, such as next-generation sequencing (NGS) and whole genome sequencing (WGS), have revolutionized the understanding of complex traits in forest trees. These technologies facilitate the identification of genetic markers, aiding breeding programmes and management strategies. Clustered regularly interspaced short palindromic repeats (CRISPR, in our study referring to CRISPR-Cas9 system) technology and the development of transgenic trees offer new possibilities for improving traits like growth, stress tolerance, and wood quality in forest species, though the potential ecological impacts warrant careful evaluation. Epigenetic research in forest trees provides insights into adaptation mechanisms to environmental changes through gene expression and phenotypic variation. The synergy between biotechnological advances and conservation practices is vital for sustaining forest ecosystems amidst rapid environmental shifts. Recommendations call for fostering interdisciplinary collaborations, enhancing biodiversity informatics infrastructure, engaging stakeholders, prioritizing epigenetic research, and developing regulatory frameworks for genome editing. These steps are imperative for a holistic approach to forest conservation, ensuring the adaptive capacity of forests and safeguarding their genetic resources against the backdrop of climate change., Klimatske promene negativno deluju na genetičke resurse širom sveta, stoga se integracija biotehnoloških inovacija sa tradicionalnim strategijama zaštite šumskih ekosistema pojavljuje kao obećavajući pravac za unapređenje otpornosti šumskih genetičkih resursa. Informatika biodiverziteta igra ključnu ulogu u upravljanju i analizi podataka, podržavajući informisano donošenje odluka u upravljanju i zaštiti šuma. Napredak u genomici i tehnologijama sekvenciranja, poput sekvenciranja sledeće generacije (NGS) i sekvenciranja celih genoma (WGS), unapredilo je razumevanje kompleksnih osobina šumskih vrsta drveća. Ove tehnologije olakšavaju identifikaciju genetičkih markera, što pomaže u procesima oplemenjivanja i strategijama upravljanja. Tehnologija grupisanih kratkih palindromskih ponovaka na jednakim rastojanjima (CRISPR, u našem radu se odnosi na CRISPR-Cas9 sistem) i razvoj transgenih individua nude nove mogućnosti za unapređenje osobina poput rasta, tolerantnosti na stres i kvaliteta drveta kod šumskih vrsta, dok potencijalni ekološki uticaji zahtevaju pažljivu evaluaciju. Epigenetička istraživanja kod šumskih vrsta drveća pružaju uvid u mehanizme adaptacije na promene u okruženju putem izražavanja gena i fenotipske varijacije. Sinergija biotehnološkog napretka i konzervacije je vitalna za održavanje šumskih ekosistema usred brzih promena u okruženju. Preporuke pozivaju na podsticanje interdisciplinarnih saradnji, unapređenje infrastrukture za informatiku biodiverziteta, uključivanje svih zainteresovanih aktera, davanje prioriteta epigenetičkim istraživanjima i razvoj regulatornih okvira za uređivanje genoma. Ovi koraci su ključni za holistički pristup očuvanju šuma, obezbeđujući njihov adaptivni kapacitet i zaštitu njihovih genetičkih resursa u svetlu klimatskih promena.

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies that we named pax37A and pax37B. The vertebrate homologs, PAX3 and PAX7 are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona intestinalis, Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found that pax37B but not pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons.

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies that we named pax37A and pax37B. The vertebrate homologs, PAX3 and PAX7 are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona intestinalis, Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found that pax37B but not pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons.

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies that we named pax37A and pax37B. The vertebrate homologs, PAX3 and PAX7 are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona intestinalis, Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found that pax37B but not pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons.

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies that we named pax37A and pax37B. The vertebrate homologs, PAX3 and PAX7 are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona intestinalis, Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found that pax37B but not pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons.

Autism Spectrum Disorders are characterized by behavioural, social, and communication impairments. Despite their prevalence, the precise mechanisms underlying ASD are poorly understood. This thesis is focused on developing suitable models to study the impact of mutations in three genes (BTBD8, FOSL2, and TAOK1) potentially associated with ASD. In vitro models were generated using human iPSCs that were differentiated into cortical neurons and human brain organoids, that were characterized through proteomics and scRNA-seq. Genes of interest were also studied in Danio rerio, to evaluate the morphological and behavioural consequences of gene dysfunction. These models will be useful for improving our understanding of the molecular mechanisms underlying ASD, but also for improving the diagnosis and treatment of the disorder.

Gene therapy is an advanced treatment approaches which are involved in insertion of genetic materials to the target cells for diseases management. It involves correcting defective genes that are appropriate for treating diseases caused by single gene failure in particularly suitable rare diseases e.g., cystic fibrosis, adrenoleukodystrophy beside liver diseases and cancers. It is looking forward to transfigure modern medicine for cure of numerous inherited metabolic liver disorders. Urgently, effective and continuous advanced biotechnology is required in developing nanoparticles vehicles, deliver mRNA biomolecules and target host cell genome. This is to reinsert the missed gene expressions, functional proteins within the target cells and disabling many obstacles. Gene editing aims to change the microorganism genetic material of DNA. It permits insertion of genetic materials, deletion, or alterations within a particular location within the gene. Many procedures have been investigated for getting gene editing. Currently, CRISPR-Cas9 is well designed DNA editing system and identified as regularly interspaced short palindromic repeats and associated with protein-9. The CRISPR-Cas9 system is cheap and fast, with high accuracy and efficiency comparing to other genetic editing tools. Molecular progress will avoid stimulating innate immunological responses. Many research studies started clinical trials based on the allowed advance in nanotechnology and molecular biology tools. They highlighted therapies to treat such liver disorders with safe and effective approaches. This study summarizes and discusses the progress in liver diseases gene therapy with a hope for success of clinical trials applications through advanced therapeutic potentials and gene editing.

The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications.

The thesis “Lipid Nanoparticle-Mediated Delivery of CRISPR-Cas9 for Therapeutic Gene Correction” delves into the potential of CRISPR-Cas9 as a gene therapy tool for treating hereditary disorders. Originally a bacterial defense mechanism, CRISPR-Cas9 has been modified into a precision gene editing tool. It functions like molecular scissors, creating specific DNA breaks that trigger DNA repair processes, which, when coupled with repair DNA, can correct gene mutations causing hereditary diseases. However, the challenge lies in safely and efficiently delivering CRISPR-Cas9 to affected organs in patients. The thesis explores the use of lipid nanoparticles, small, virus-sized particles composed of naturally occurring body fats, as a viable delivery system for CRISPR-Cas9. The work in this thesis demonstrates that the designed lipid nanoparticles can effectively transport CRISPR-Cas9 into both cultured cells and later in liver cells of mice as well. Moreover, the results show that the delivered CRISPR-Cas9 generates precise genome edits. Furthermore, it addresses the potential immune responses against CRISPR-Cas9, given it bacterial origin, and introduces a strategy to mitigate such reactions. In summary, this thesis presents a promising approach to deliver CRISPR-Cas9 to diseased cells, enabling the precise correction of pathogenic gene mutations, marking a significant advancement in the field of gene therapy for hereditary disorders.

Advances in cell-based therapy, particularly CAR-T cell therapy, have transformed the treatment of hematological malignancies. Although an important step forward for the field, autologous CAR-T therapies are hindered by high costs, manufacturing challenges, and limited efficacy against solid tumors. With ongoing progress in gene editing and culture techniques, engineered stem cells and their application in cell therapy are poised to address some of these challenges. Here, we review stem cell-based immunotherapy approaches, stem cell sources, gene engineering and manufacturing strategies, therapeutic platforms, and clinical trials, as well as challenges and future directions for the field.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

Background: Multiple surveys of the general public and experts on human genome editing have been conducted. However, many focused only on editing in clinical applications, with few regarding its use for basic research. Given that genome editing for research purposes is indispensable for the realization of clinical genome editing, understanding lay attitudes toward genome editing in research, particularly using human embryos, which is likely to provoke ethical concerns, is helpful for future societal discussion. Methods: An online survey was conducted with Japanese laypeople and researchers to ascertain their views regarding human genome editing for research purposes. Participants were queried about their acceptance as a function of the target of genome editing (germ cells, surplus IVF embryos, research embryos, somatic cells); then, those who answered “acceptable depending on the purpose” were asked about their acceptance in the context of specific research purposes of genome editing. Participants were also asked about their expectations and concerns regarding human genome editing. Results: Replies were obtained from 4, 424 laypeople and 98 researchers. Approximately 28.2–36.9% of the laypeople exhibited strong resistance to genome editing for research purposes regardless of their applications. In contrast, 25.5% of the researchers demonstrated resistance only to genome editing in research embryos; this percentage was substantially higher than those concerning the other three targets (5.1–9.2%). Approximately 50.4–63.4% of laypeople who answered “acceptable depending on the purpose” approved germline genome editing for disease research; however, only 39.3–42.8% approved genome editing in basic research to obtain biological knowledge. In contrast, the researchers displayed a lower degree of acceptance of germline genome editing for research purposes related to chronic diseases (60.9–66.7%) than for other research purposes (73.6–90.8%). Analysis of responses concernin

Maligna cancerceller har förmågan att sprida sig okontrollerat i kroppen och metastasera till avlägsna organ. Kritiskt i metastaserings processen är cancercellers förmåga att invadera närliggande vävnader innan de sprider sig vidare ut i kroppen. Att ha en förståelse för mekanismerna som bidrar till cancercellers invasiva egenskaperna kan därför leda till upptäckten av nya läkemedel som kan förhindra metastaser. Den här studien undersökte därför de invasiva egenskaperna hos humana melanomceller in vitro med en CRISPR knockout screen. Först utvärderades tre poolade CRISPR bibliotek, där ett av dem valdes ut till en slutgiltig screen. När gRNA representationen för det utvalda epigentiska knockout biblioteket hade validerats, genererades ett lentiviralt bibliotek för transducering av A375 melanomceller. Förmågan hos melanomcellerna att invadera genom epitelmembran undersöktes sedan med en optimerad invasionassay med Matrigel invasion chambers. Transducerade melanoma celler tillsattes till den övre kammaren och fick migrera genom Matrigelen mot den nedre kammaren som hade en högre koncentration av kemoattraktant. Därefter extraherades celler från den övre och nedre kammaren separat och deras genomiska DNA isolerades. Sekvenseringsbiblioteket genererades med PCR och sekvenserades med Illumina next-generation sekvenserings teknologi. Denna rapport innehåller dock inte sekvenseringsdata från en CRISPR knockout screen., Malignant cancer cells undergo uncontrolled proliferation and can metastasize to distant organs. A critical step in metastasis is the ability of cancer cells to invade neighboring tissues before they can disseminate to distant organs. Understanding the mechanisms of invasiveness could therefore lead to the discovery of new druggable targets to prevent metastasis. In this project, the invasiveness properties of human melanoma cells were studied in vitro using a CRISPR knockout screen. For this, three pooled CRISPR libraries were evaluated, and then one of them was selected for the screen. When the gRNA representation of the selected epigentic knockout library had been validated, a lentiviral library was generated for transduction of A375 melanoma cells. The invasiveness of the mutated melanoma cells was then examined with an optimized invasion assay using Matrigel invasion chambers. Transduced melanoma cells were seeded into the upper chamber and allowed to migrate through the Matrigel towards the bottom chamber that had a higher concentration of chemoattractant. Cells in the upper and bottom chamber were then separately collected and their genomic DNA was isolated. The sequencing library was produced by PCR amplification and sequenced with Illumina next-generation sequencing technology. The sequencing data from the CRISPR screen is not included in this report.

Strategic management is crucial for organizational success in today's volatile business environment, and yet, underutilized within agricultural businesses. The agricultural industry is facing rapid change with the introduction of new GM technologies, such as CRISPR-Cas9, and following regulations. Although these technologies have the potential to provide farmers with improved crops containing desired traits, excessive regulations and obstacles hinder its implementation in European agricultural practices. If the upcoming decision regarding CRISPR-Cas9 allows the technology to not be treated under GMO regulations, European agriculture is faced with a major opportunity. However, as procedures are unexplored, organizations are left unprepared. This study investigates the optimal strategic planning approach for Swedish agricultural businesses wanting to efficiently implement new technologies and adjust to regulatory changes. The research is based on a qualitative study that incorporates deductive methodology. It follows a multiple case study research design with eight semi-structured interviews from six Swedish and American case companies as the primary data collection method. By taking inspiration from experienced companies’ planning strategies and including European values, Swedish agricultural companies wanting to implement CRISPR-Cas9 can efficiently navigate changes within the industry while maintaining a competitive advantage. A simple model combining parts from short- and long-term planning frameworks with communication and value creation can guide companies entering the upcoming field.

Genome editing is an exciting field that allows for the precise modification of an organism's DNA. One of the most advanced tools in this area is CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), which creates a DSB (Double-strand break) at a specific location in the genome. This break can then be repaired by the cell using one of two pathways – NHEJ (nonhomologous end joining) or HDR (homology-directed repair) HDR leads to more precise repair and is used to create KI (Knock-In) modifications by introducing a homologous piece of DNA with the desired changes. However, HDR is a rare event that competes with the error prone NHEJ pathway, limiting its efficiency. HDR mainly occurs in the G2 and S phases of the cell cycle, making it a challenge to control and target. To improve KI efficiency, researchers have used strategies such as inhibiting NHEJ or activating HDR. This study focuses on identifying direct and indirect activators of HDR through a library assay screening. We established a robust method for screening compounds in HEK293 cells that relies on a plasmid-based delivery Cas9, gRNA (guide RNA), and synthetic ssDNA (single strand DNA). Out of 3,000 compounds screened, 1% showed a higher signal than the positive control, and approximately 10% presented a higher signal than untreated cells. The top 5 compounds were further validated in dose response. Our system opens new avenues for improving the efficiency of KI modifications.

It is widely accepted that aggressive development of the bio-economy is needed to mitigate the effects of imminent resource shortages and climate change. The employment of microorganisms, such as yeasts, as biorefineries is one feasible route to meet this demand. Kluyveromyces marxianus is a non-conventional yeast commonly found in fermented dairy products that is exceptionally fast-growing, thermo- and pH-tolerant, metabolizes a wide range of low-cost carbon sources, and has great potential for biosynthesis of acetyl-CoA-based products. In this work we demonstrate the high native capacity of K. marxianus to produce triacetic acid lactone (TAL), a member of the diverse polyketide class of bioactive molecules, from waste substrates, achieving a yield comparable to other industrially relevant yeasts that required extensive engineering. Despite these advantageous characteristics, K. marxianus has not been widely engineered; this is due in part to the strong non-homologous end joining pathway which greatly hinders targeted genomic modification. The recent emergence of CRISPR-Cas9 genome editing technology has alleviated this roadblock, allowing metabolic engineering of K. marxianus to be more accessible. By constructing a highly efficient CRISPR-Cas9 system and disrupting the non-homologous end joining pathway, we have created a robust and efficient platform that routinely enables a high rate of targeted editing in K. marxianus, including genomic integrations with as little as 40 base pair homologies. Using this tool, we have conducted rationally and computationally guided metabolic engineering to significantly improve the biosynthesis of TAL and an additional polyketide, 6-methylsalisylic acid (6-MSA). Combining the best performing modifications led to 1.66- and 2.5-fold increases in TAL and 6-MSA biosynthesis, respectively, over the unengineered strains in defined media containing xylose or lactose. Altogether, our work demonstrates effective strategies for targeted m

Proposed genetic approaches for reducing human malaria include population modification, which introduces genes into vector mosquitoes to reduce or prevent parasite transmission. We demonstrate the potential of Cas9/guide RNA (gRNA)-based gene-drive systems linked to dual antiparasite effector genes to spread rapidly through mosquito populations. Two strains have an autonomous gene-drive system coupled to dual anti-Plasmodium falciparum effector genes comprising single-chain variable fragment monoclonal antibodies targeting parasite ookinetes and sporozoites in the African malaria mosquitoes Anopheles gambiae (AgTP13) and Anopheles coluzzii (AcTP13). The gene-drive systems achieved full introduction within 3 to 6 mo after release in small cage trials. Life-table analyses revealed no fitness loads affecting AcTP13 gene-drive dynamics but AgTP13 males were less competitive than wild types. The effector molecules reduced significantly both parasite prevalence and infection intensities. These data supported transmission modeling of conceptual field releases in an island setting that shows meaningful epidemiological impacts at different sporozoite threshold levels (2.5 to 10 k) for human infection by reducing malaria incidence in optimal simulations by 50 to 90% within as few as 1 to 2 mo after a series of releases, and by ≥90% within 3 mo. Modeling outcomes for low sporozoite thresholds are sensitive to gene-drive system fitness loads, gametocytemia infection intensities during parasite challenges, and the formation of potentially drive-resistant genome target sites, extending the predicted times to achieve reduced incidence. TP13-based strains could be effective for malaria control strategies following validation of sporozoite transmission threshold numbers and testing field-derived parasite strains. These or similar strains are viable candidates for future field trials in a malaria-endemic region.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently

Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistant Candida auris have been reported worldwide. Although hotspot mutations in FKS1 are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced the FKS1 gene of a caspofungin-resistant clinical isolate ( clade I ) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clus-tered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced into C. auris wild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to the FKS1 R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance in C. auris., Journal of Fungi, 9(5), art. no. 529; 2023

Genome editing is an exciting field that allows for the precise modification of an organism's DNA. One of the most advanced tools in this area is CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), which creates a DSB (Double-strand break) at a specific location in the genome. This break can then be repaired by the cell using one of two pathways – NHEJ (nonhomologous end joining) or HDR (homology-directed repair) HDR leads to more precise repair and is used to create KI (Knock-In) modifications by introducing a homologous piece of DNA with the desired changes. However, HDR is a rare event that competes with the error prone NHEJ pathway, limiting its efficiency. HDR mainly occurs in the G2 and S phases of the cell cycle, making it a challenge to control and target. To improve KI efficiency, researchers have used strategies such as inhibiting NHEJ or activating HDR. This study focuses on identifying direct and indirect activators of HDR through a library assay screening. We established a robust method for screening compounds in HEK293 cells that relies on a plasmid-based delivery Cas9, gRNA (guide RNA), and synthetic ssDNA (single strand DNA). Out of 3,000 compounds screened, 1% showed a higher signal than the positive control, and approximately 10% presented a higher signal than untreated cells. The top 5 compounds were further validated in dose response. Our system opens new avenues for improving the efficiency of KI modifications.

Medical enhancement), a step close to transhumanism, has positioned itself in our society. Transhumanism aims to use science and technology to take human beings beyond the limitations of their natural form, with the intention of subsequently moving to posthumanism, the creation of a new species. Recent scientific advances, especially in the genetic (field, may make these options possible. Electromechanical innovations, such as the miniaturization of components and the improvement of biocompatible materials, have led to advances in brain-machine interfaces that enhance a cybernetic dimension, in which mechanical devices would be under the direct control of the mind. The field of health sciences is particularly fruitful for the application of these technologies, leading to improvements in the human being, which can be applied to the medical enhancement and transhumanism. This reflection offers an overview of the most important of these recent advances, with special emphasis on genome editing. Issues of posthumanism will not be specifically considered., La medicina del mejoramiento es un paso cercano al transhumanismo y se ha posicionado en nuestra sociedad. Este movimiento cultural, pretende utilizar la ciencia y la tecnología para llevar al ser humano más allá de las limitaciones de su forma natural, con la intención de posteriormente pasar al posthumanismo, la creación de una nueva especie. Los últimos avances científicos, especialmente en el campo genético, pueden posibilitar estas opciones. Las innovaciones electromecánicas, como la miniaturización de los componentes y la mejora de los materiales biocompatibles, han propiciado avances en las interfaces cerebro-máquina que potencian una dimensión cibernética, en la que los dispositivos mecánicos estarían bajo el control directo de la mente. El campo de las ciencias de la salud, es especialmente fructífero para la aplicación de estas tecnologías, propiciando en el ser humano mejoras, que pueden ser aplicadas a la medicina del mejoramiento y al transhumanismo. Esta reflexión ofrece una visión de los más importantes de estos avances recientes, con especial énfasis en la edición del genoma humano. No se considerarán en esta contribución específicamente temas de posthumanismo.

Free sialic acid storage disorders (FSASDs) result from pathogenic variations in the SLC17A5 gene, which encodes the lysosomal transmembrane protein sialin. Loss or deficiency of sialin impairs FSA transport out of the lysosome, leading to cellular dysfunction and neurological impairment, with the most severe form of FSASD resulting in death during early childhood. There are currently no therapies for FSASDs. Here, we evaluated the efficacy of CRISPR-Cas9-mediated homology directed repair (HDR) and adenine base editing (ABE) targeting the founder variant, SLC17A5 c.115C>T (p.Arg39Cys) in human dermal fibroblasts. We observed minimal correction of the pathogenic variant in HDR samples with a high frequency of undesired insertions/deletions (indels) and significant levels of correction for ABE-treated samples with no detectable indels, supporting previous work showing that CRISPR-Cas9-mediated ABE outperforms HDR. Furthermore, ABE treatment of either homozygous or compound heterozygous SLC17A5 c.115C>T human dermal fibroblasts demonstrated significant FSA reduction, supporting amelioration of disease pathology. Translation of this ABE strategy to mouse embryonic fibroblasts harboring the Slc17a5 c.115C>T variant in homozygosity recapitulated these results. Our study demonstrates the feasibility of base editing as a therapeutic approach for the FSASD variant SLC17A5 c.115C>T and highlights the usefulness of base editing in monogenic diseases where transmembrane protein function is impaired.

Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistant Candida auris have been reported worldwide. Although hotspot mutations in FKS1 are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced the FKS1 gene of a caspofungin-resistant clinical isolate ( clade I ) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clus-tered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced into C. auris wild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to the FKS1 R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance in C. auris., Journal of Fungi, 9(5), art. no. 529; 2023

Sesquiterpene lactones (STL) are a group of natural compounds found in various plant species, including sunflowers (Helianthus spp.), and they have been studied for their potential role in allelopathy and defense against pests and pathogens. Allelopathy refers to the ability of certain plants to release chemicals that affect the growth and development of neighboring plants and organisms. In the context of sunflower broomrape (Orobanche cumana) and sunflowers, STLs have been of particular interest due to their potential role in inhibiting the growth of the broomrape parasite. Some STLs have been shown to possess allelopathic properties, which means they can influence the germination and growth of other plants, potentially including parasitic plants like sunflower broomrape. Research has suggested that certain STLs found in sunflowers may exhibit inhibitory effects on the germination and growth of sunflower broomrape seeds. These compounds could potentially be released from the sunflower roots and into the soil, creating a hostile environment for the parasite. However, the effectiveness of sesquiterpene lactones in controlling sunflower broomrape is still an active area of research, and their practical application as a management strategy requires further investigation.

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.

CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89 g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing. Algorithms for the prediction of efficacy of the sgRNAs had little predictive power in this system. The analysis of mutations suggested predictable identity of single base insertions. Off-target mutations were found for 13 out of 89 sgRNAs and only occurred at positions with one or two mismatches (at 14 and 3 sites, respectively). We found that PAM-proximal mismatches do not preclude low frequency off-target mutations. Off-target mutations were not found at all 138 positions that had three or four mismatches. We compared off-target mutation frequencies obtained with plasmid encoding sgRNAs and Cas9 with those induced by ribonucleoprotein (RNP) transfections. The use of RNPs led to a significant decrease in relative off-target frequencies at 6 out of 17, no significant difference at 9, and an increase at 2 sites. Additionally, we show that off-target sequences with insertions or deletions relative to the sgRNA may be mutated, and should be considered during sgRNA design. Altogether, our data help sgRNA design by providing insight into the Cas9-induced double-strand break repair outcomes and the occurrence of off-target mutations.

Sunflower is one of the most important oil crops in the world that become a strategic plant due to the increased demand for its oil in recent years. Sunflower has low climate demand that enables it to grow in many regions of Europe. The biggest problem in sunflower cultivation is the presence of parasitic plants called broomrape (O. cumana). The seeds of these non-photosynthetic parasitic plants germinate with the secretion of Sesquiterpene Lactones (STL) from the roots of the sunflower. After attachment to the roots of the sunflower, it absorbs the water and all the nutrients from the host. Just one broomrape plant can produce millions of tiny seeds that can survive more than 15 years in the soil and can contaminate many fields in a region. Classical herbicides and mechanical techniques are not effective on these parasitic plants, since it already causes great damage to the plant when it rises above the ground. Many sunflower lines resistant to broomrape have been developed in last decades. However, the resistance of these lines was broken by emergence of new virulent broomrape strains. In recent years, secretion of Sesquiterpene Lactones (STLs) from sunflower roots has been found to trigger the germination of broomrape seeds. The genes encoding the enzymes (HaGAS, HaGAO, HaG8H, HaCOS) functional in STL biosynthesis in sunflower have been well characterized in recent years. In the light of all these information, genes of the enzymes that catalyze the production STLS was aimed to knockout with CRISPR/Cas9 technique in the project.

CRISPR-Cas9 has emerged as a revolutionary tool that enables precise and efficient modifications of the genetic material. This review provides a comprehensive overview of CRISPR-Cas9 technology and its applications in genome editing. We begin by describing the fundamental principles of CRISPR-Cas9 technology, explaining how the system utilizes a single guide RNA (sgRNA) to direct the Cas9 nuclease to specific DNA sequences in the genome, resulting in targeted double-stranded breaks. In this review, we provide in-depth explorations of CRISPR-Cas9 technology and its applications in agriculture, medicine, environmental sciences, fisheries, nanotechnology, bioinformatics, and biotechnology. We also highlight its potential, ongoing research, and the ethical considerations and controversies surrounding its use. This review might contribute to the understanding of CRISPR-Cas9 technology and its implications in various fields, paving the way for future developments and responsible applications of this transformative technology.

Strategic management is crucial for organizational success in today's volatile business environment, and yet, underutilized within agricultural businesses. The agricultural industry is facing rapid change with the introduction of new GM technologies, such as CRISPR-Cas9, and following regulations. Although these technologies have the potential to provide farmers with improved crops containing desired traits, excessive regulations and obstacles hinder its implementation in European agricultural practices. If the upcoming decision regarding CRISPR-Cas9 allows the technology to not be treated under GMO regulations, European agriculture is faced with a major opportunity. However, as procedures are unexplored, organizations are left unprepared. This study investigates the optimal strategic planning approach for Swedish agricultural businesses wanting to efficiently implement new technologies and adjust to regulatory changes. The research is based on a qualitative study that incorporates deductive methodology. It follows a multiple case study research design with eight semi-structured interviews from six Swedish and American case companies as the primary data collection method. By taking inspiration from experienced companies’ planning strategies and including European values, Swedish agricultural companies wanting to implement CRISPR-Cas9 can efficiently navigate changes within the industry while maintaining a competitive advantage. A simple model combining parts from short- and long-term planning frameworks with communication and value creation can guide companies entering the upcoming field.

Eriodictyol is a hydroxylated flavonoid displaying multiple pharmaceutical activities, such as antitumoral, antiviral or neuroprotective. However, its industrial production is limited to extraction from plants due to its inherent limitations. Here, we present the generation of a Streptomyces albidoflavus bacterial factory edited at the genome level for an optimized de novo heterologous production of eriodictyol. For this purpose, an expansion of the Golden Standard toolkit (a Type IIS assembly method based on the Standard European Vector Architecture (SEVA)) has been created, encompassing a collection of synthetic biology modular vectors (adapted for their use in actinomycetes). These vectors have been designed for the assembly of transcriptional units and gene circuits in a plug-and-play manner, as well as for genome editing using CRISPR-Cas9-mediated genetic engineering. These vectors have been used for the optimization of the eriodictyol heterologous production levels in S. albidoflavus by enhancing the flavonoid-3′-hydroxylase (F3’H) activity (by means of a chimera design) and by replacing three native biosynthetic gene clusters in the bacterial chromosome with the plant genes matBC (involved in extracellular malonate uptake and its intracellular activation into malonyl-CoA), therefore allowing more malonyl-CoA to be devoted to the heterologous production of plant flavonoids in this bacterial factory. These experiments have allowed an increase in production of 1.8 times in the edited strain (where the three native biosynthetic gene clusters have been deleted) in comparison with the wild-type strain and a 13 times increase in eriodictyol overproduction in comparison with the non-chimaera version of the F3′H enzyme.

DNA sequence variants (single nucleotide polymorphisms or variants, SNPs/SNVs; copy number variants, CNVs) associated to neurodevelopmental disorders (NDD) and traits often map on putative transcriptional regulatory elements, including, in particular, enhancers. However, the genes controlled by these enhancers remain poorly defined. Traditionally, the activity of a given enhancer, and the effect of its possible alteration associated to the sequence variants, has been thought to influence the nearest gene promoter. However, the obtainment of genome-wide long-range interaction maps in neural cells chromatin challenged this view, showing that a given enhancer is very frequently not connected to the nearest promoter, but to a more distant one, skipping genes in between. In this Perspective, we review some recent papers, who generated long-range interaction maps (by HiC, RNApolII ChIA-PET, Capture-HiC, or PLACseq), and overlapped the identified long-range interacting DNA segments with DNA sequence variants associated to NDD (such as schizophrenia, bipolar disorder and autism) and traits (intelligence). This strategy allowed to attribute the function of enhancers, hosting the NDD-related sequence variants, to a connected gene promoter lying far away on the linear chromosome map. Some of these enhancer-connected genes had indeed been already identified as contributive to the diseases, by the identification of mutations within the gene’s protein-coding regions (exons), validating the approach. Significantly, however, the connected genes also include many genes that were not previously found mutated in their exons, pointing to novel candidate contributors to NDD and traits. Thus, long-range interaction maps, in combination with DNA variants detected in association with NDD, can be used as “pointers” to identify novel candidate disease-relevant genes. Functional manipulation of the long-range interaction network involving enhancers and promoters by CRISPR-Cas9-based approache

CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted double-strand breaks are controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming, and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome editing in S. cerevisiae, pCEC-red. This tool allows to (i) transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and (ii) insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and (iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (∼90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.

Accurate genome editing based on various molecular tools has always been the focus of gene-editing research and the primary goal for therapeutic application. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a well-established gene-editing method that is preferred due to its simplicity and high efficiency. In this study, a group of single-stranded DNA aptamers with high affinity and high specificity for the Cas9 protein were obtained by the systematic evolution of ligands through the exponential enrichment method. Their binding affinity and possible binding domains to the Cas9 protein were analyzed. In addition, we demonstrated the effectiveness of aptamers in regulating dCas9-modulated gene transcription, in terms of both transcriptional activation and repression. Additionally, the aptamers successfully reduced the off-target effect and improved the efficiency of gene homologous recombination repair mediated by CRISPR-Cas9. The findings suggest a potential method to better control precise gene editing and enrich the diversity of modulating tools for the CRISPR-Cas9 system. © 2022 The Author(s)

Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated. The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.

Maligna cancerceller har förmågan att sprida sig okontrollerat i kroppen och metastasera till avlägsna organ. Kritiskt i metastaserings processen är cancercellers förmåga att invadera närliggande vävnader innan de sprider sig vidare ut i kroppen. Att ha en förståelse för mekanismerna som bidrar till cancercellers invasiva egenskaperna kan därför leda till upptäckten av nya läkemedel som kan förhindra metastaser. Den här studien undersökte därför de invasiva egenskaperna hos humana melanomceller in vitro med en CRISPR knockout screen. Först utvärderades tre poolade CRISPR bibliotek, där ett av dem valdes ut till en slutgiltig screen. När gRNA representationen för det utvalda epigentiska knockout biblioteket hade validerats, genererades ett lentiviralt bibliotek för transducering av A375 melanomceller. Förmågan hos melanomcellerna att invadera genom epitelmembran undersöktes sedan med en optimerad invasionassay med Matrigel invasion chambers. Transducerade melanoma celler tillsattes till den övre kammaren och fick migrera genom Matrigelen mot den nedre kammaren som hade en högre koncentration av kemoattraktant. Därefter extraherades celler från den övre och nedre kammaren separat och deras genomiska DNA isolerades. Sekvenseringsbiblioteket genererades med PCR och sekvenserades med Illumina next-generation sekvenserings teknologi. Denna rapport innehåller dock inte sekvenseringsdata från en CRISPR knockout screen., Malignant cancer cells undergo uncontrolled proliferation and can metastasize to distant organs. A critical step in metastasis is the ability of cancer cells to invade neighboring tissues before they can disseminate to distant organs. Understanding the mechanisms of invasiveness could therefore lead to the discovery of new druggable targets to prevent metastasis. In this project, the invasiveness properties of human melanoma cells were studied in vitro using a CRISPR knockout screen. For this, three pooled CRISPR libraries were evaluated, and then one of them was selected for the screen. When the gRNA representation of the selected epigentic knockout library had been validated, a lentiviral library was generated for transduction of A375 melanoma cells. The invasiveness of the mutated melanoma cells was then examined with an optimized invasion assay using Matrigel invasion chambers. Transduced melanoma cells were seeded into the upper chamber and allowed to migrate through the Matrigel towards the bottom chamber that had a higher concentration of chemoattractant. Cells in the upper and bottom chamber were then separately collected and their genomic DNA was isolated. The sequencing library was produced by PCR amplification and sequenced with Illumina next-generation sequencing technology. The sequencing data from the CRISPR screen is not included in this report.

In this study, we review the methodology used to develop a molecular marker for fruit flesh color in Japanese plum, a species in which marker-assisted breeding is still in its infancy. We focus on the use and potential of the CRISPR-Cas9 enrichment sequencing, a novel methodology that attains the long-read target sequencing of genomic regions of interest. This technology allowed the sequencing of the highly polymorphic and duplicated LG3-MYB10 region of five plum cultivars in a cost-effective manner. This resulted in the identification of a structural variant associated with flesh color, which was an LTR retrotransposon of 2.8 kb inserted in the promoter of the PsMYB10.2 gene and allowed the design of an efficient molecular marker for the trait. The role of the PsMYB10.2 gene as an inducer of anthocyanin biosynthesis in the flesh was validated by transient overexpression in Japanese plum fruits. The long-terminal repeat retrotransposon might have a role in gene activation, as reported in other species. The study reviewed here is a great example that highlights the potential of CRISPR-Cas9 enrichment sequencing as a tool to identify genetic variations in genomic regions of interest, even if these are highly complex. As with Japanese plum and flesh color, this methodology could be a powerful tool for the development of molecular markers linked to traits of interest and agronomic value in any species.

Autosomal recessive limb-girdle muscular dystrophy 21 (LGMDR21) is caused by pathogenic variants in protein O-glucosyltransferase 1 (POGLUT1), which is responsible for O-glucosylation of specific epidermal growth factor (EGF) repeats found in ∼50 mammalian proteins, including Notch receptors. Previous data from patient biopsies indicated that impaired Notch signaling, reduction of muscle stem cells, and accelerated differentiation are probably involved in disease etiopathology. Using patient induced pluripotent stem cells (iPSCs), their corrected isotypes, and control iPSCs, gene expression profiling indicated dysregulation of POGLUT1, NOTCH, muscle development, extracellular matrix (ECM), cell adhesion, and migration as involved pathways. They also exhibited reduced in vitro POGLUT1 enzymatic activity and NOTCH signaling as well as defective myogenesis, proliferation, migration and differentiation. Furthermore, in vivo studies demonstrated significant reductions in engraftment, muscle stem cell formation, PAX7 expression, and maintenance, along with an increased percentage of mislocalized PAX7+ cells in the interstitial space. Gene correction in patient iPSCs using CRISPR-Cas9 nickase led to the rescue of the main in vitro and in vivo phenotypes. These results demonstrate the efficacy of iPSCs and gene correction in disease modeling and rescue of the phenotypes and provide evidence of the involvement of muscle stem cell niche localization, PAX7 expression, and cell migration as possible mechanisms in LGMDR21.

The current great extinction of species is due to the destruction of habitats and ecosystems. Covid-19 revealed false assurances; believe that it is enough to improve the existing rules is denying reality. Earth system sciences teach us that we are hyperconnected and the new biology highlights the relevance of the horizontal transmission of information from «genetic libraries.» Evolution is not «random mutations» of individuals, but is due to symbiosis between different kingdoms. The conservation of individuals, without understanding the complexity of the biosphere, offers disappointing results. There are voices that question preserving biodiversity by getting rid of «genetic options libraries» by eliminating «junk DNA» and with a massive extinction of «invasive exotic species». The scientific community is excited about gene editing: it promises everything from biological «functional gains» to preserving biodiversity. It does not consider the role of «recessive genes» in «random mutations», preferring the homogeneity of genes than an endless chain of interactions. In the article we expressed surprise that CRISPR technology impoverishes the genetic pool accumulated over millions of years., La actual gran extinción de especies se debe a la destrucción de hábitats y ecosistemas. El COVID-19 puso de manifiesto falsas seguridades; creer que basta mejorar las reglas existentes es negar la realidad. Las ciencias del sistema terrestre nos enseñan que estamos hiperconectados, y la nueva biología destaca la relevancia de la transmisión horizontal de información de las «bibliotecas genéticas». La evolución no se basa en «mutaciones aleatorias» de individuos, sino que se debe a la simbiosis entre distintos reinos. La conservación de individuos sin entender la complejidad de la biosfera ofrece resultados decepcionantes. Hay voces que cuestionan que se preserve la biodiversidad si nos deshacemos de «bibliotecas de opciones genéticas», eliminamos el «ADN basura» y provocamos una masiva extinción de «especies exóticas invasoras». La comunidad científica está entusiasmada con la edición genética: promete desde «ganancias funcionales» biológicas hasta preservar la biodiversidad. No tiene en cuenta el papel de «genes recesivos» en «mutaciones aleatorias», al preferir la homogeneidad de genes a una cadena interminable de interacciones. Expresamos en el artículo estupor que la tecnología CRISPR, que empobrece el acervo genético acumulado durante millones de años.

Introduction: Most lung diseases are serious conditions resulting from genetic and environmental causes associated with high mortality and severe symptoms. Currently, treatments available have a palliative effect and many targets are still considered undruggable. Gene therapy stands as an attractive approach to offering innovative therapeutic solutions. CRISPRCas9 has established a remarkable potential for genome editing with high selectivity to targeted mutations. To ensure high efficacy with minimum systemic exposure, the delivery and administration route are key components that must be investigated. Areas covered: This review is focused on the delivery of CRISPRCas9 to the lungs, taking advantage of lipid nanoparticles (LNPs), the most clinically advanced nucleic acid carriers. We also aim to highlight the benefits of pulmonary administration as a local delivery route and the use of spray drying to prepare stable nucleic-acid-based dry powder formulations that can overcome multiple lung barriers. Expert opinion: Exploring the pulmonary administration to deliver CRISPRCas9 loaded in LNPs as a dry powder increases the chances to achieve high efficacy and reduced adverse effects. CRISPRCas9 loaded in LNP-embedded microparticles has not yet been reported in the literature but has the potential to reach and accumulate in target cells in the lung, thus, enhancing overall efficacy and safety.

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Current approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed “direct parental” CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemocoel of adult females efficiently introduces heritable mutations in developing oocytes. Importantly, commercially available standard Cas9 protein can be directly used for DIPA-CRISPR, which makes this approach highly practical and feasible. DIPA-CRISPR enables highly efficient gene editing in the cockroaches, on which conventional approaches cannot be applied, and in the model beetle Tribolium castaneum. Due to its simplicity and accessibility, DIPA-CRISPR will greatly extend the application of gene editing technology to a wide variety of insects.

Recent advances in genetics, molecular biology, and stem cell biology have accelerated our understanding of neuropsychiatric disorders, like autism spectrum disorder (ASD), major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SZ). This progress highlights the incredible complexity of both the human brain and mental illnesses from the biochemical to the cellular level. Contributing to the complexity of neuropsychiatric disorders are their polygenic nature, cellular and brain region interconnectivity, and dysregulation of human-specific neurodevelopmental processes. Here, we discuss available tools, including CRISPR-Cas9, and the applications of these tools to develop cell-based two-dimensional (2D) models and 3D brain organoid models that better represent and unravel the intricacies of neuropsychiatric disorder pathophysiology.