AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

Genome Editing Dilemma: Navigating Dual-Use Potential and Charting the Path Forward.

Contemporary genome editing techniques have made genomic intervention-from microorganism to human-more accessible, easier to use, and more accurate than previous methods. We argue that, notwithstanding its merits in treating and preventing disease in humans, genome editing represents a potential threat for domestic and international security, requiring an integrated approach in regulating, detecting, preventing, and mitigating the risk of its use for malicious purposes. Despite the global regulatory ambitions of the 2021 WHO framework, we see insufficient attention given to the future prospect of dual-use genomic technology. Drawing parallels with the nuclear field, we suggest tentative practical steps for a way forward in dealing with genome editing technologies, such as: 1) adapting national (bio)security and defence strategies to include genome editing as a possible threat (with conceivable WMD potential); 2) enhancing the international dialogue on genome editing and raising the issue at the highest level; 3) working towards a global, legally binding verification mechanism; 4) tracking genome editing technologies.

CRISPR/Cas9-Editing K562 Cell Line as a Potential Tool in Transfusion Applications: Knockout of Vel Antigen Gene.

Introduction: The Vel- phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin.

Development of a live attenuated vaccine candidate for equid alphaherpesvirus 1 control: a step towards efficient protection.

Equid alphaherpesvirus 1 (EqAHV1) is a viral pathogen known to cause respiratory disease, neurologic syndromes, and abortion storms in horses. Currently, there are no vaccines that provide complete protection against EqAHV1. Marker vaccines and the differentiation of infected and vaccinated animals (DIVA) strategy are effective for preventing and controlling outbreaks but have not been used for the prevention of EqAHV1 infection. Glycoprotein 2 (gp2), located on the envelope of viruses (EqAHV1), exhibits high antigenicity and functions as a molecular marker for DIVA. In this study, a series of EqAHV1 mutants with deletion of gp2 along with other virulence genes (TK, UL24/TK, gI/gE) were engineered. The mutant viruses were studied in vitro and then in an in vivo experiment using Golden Syrian hamsters to assess the extent of viral attenuation and the immune response elicited by the mutant viruses in comparison to the wild-type (WT) virus. Compared with the WT strain, the YM2019 Δgp2, ΔTK/gp2, and ΔUL24/TK/gp2 strains exhibited reduced growth in RK-13 cells, while the ΔgI/gE/gp2 strain exhibited significantly impaired proliferation. The YM2019 Δgp2 strain induced clinical signs and mortality in hamsters. In contrast, the YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 variants displayed diminished pathogenicity, causing no observable clinical signs or fatalities. Immunization with nasal vaccines containing YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 elicited a robust immune response in hamsters. In particular, compared with the vaccine containing the ΔTK/gp2 strain, the vaccine containing the ΔUL24/TK/gp2 strain demonstrated enhanced immune protection upon challenge with the WT virus. Furthermore, an ELISA for gp2 was established and refined to accurately differentiate between infected and vaccinated animals. These results confirm that the ΔUL24/TK/gp2 strain is a safe and effective live attenuated vaccine candidate for controlling EqAHV1 infection.

Elucidating the mechano-molecular dynamics of TRAP activity using CRISPR/Cas9 mediated fluorescent reporter mice.

Osteoclasts are essential for bone remodeling by adapting their resorptive activity in response to their mechanical in vivo environment. However, the molecular mechanisms underlying this process remain unclear. Here, we demonstrated the role of tartrate-resistant acid phosphatase (TRAP, Acp5), a key enzyme secreted by osteoclasts, in bone remodeling and mechanosensitivity. Using CRISPR/Cas9 reporter mice, we demonstrated bone cell reporter (BCRIbsp/Acp5) mice feature fluorescent TRAP-deficient osteoclasts and examined their activity during mechanically driven trabecular bone remodeling. Although BCRIbsp/Acp5 mice exhibited trabecular bone impairments and reduced resorption capacity in vitro, RNA sequencing revealed unchanged levels of key osteoclast-associated genes such as Ctsk, Mmp9, and Calcr. These findings, in conjunction with serum carboxy-terminal collagen crosslinks (CTX) and in vivo mechanical loading outcomes collectively indicated an unaltered bone resorption capacity of osteoclasts in vivo. Furthermore, we demonstrated similar mechanoregulation during trabecular bone remodeling in BCRIbsp/Acp5 and wild-type (WT) mice. Hence, this study provides valuable insights into the dynamics of TRAP activity in the context of bone remodeling and mechanosensation.

Exploring the De Novo NMN Biosynthesis as an Alternative Pathway to Enhance NMN Production.

Nicotinamide mononucleotide (NMN) serves as a precursor for NAD+ synthesis and has been shown to have positive effects on the human body. Previous research has predominantly focused on the nicotinamide phosphoribosyltransferase-mediated route (NadV-mediated route) for NMN biosynthesis. In this study, we have explored the de novo NMN biosynthesis route as an alternative pathway to enhance NMN production. Initially, we systematically engineered Escherichia coli to enhance its capacity for NMN synthesis and accumulation, resulting in a remarkable over 100-fold increase in NMN yield. Subsequently, we progressively enhanced the de novo NMN biosynthesis route to further augment NMN production. We screened and identified the crucial role of MazG in catalyzing the enzymatic cleavage of NAD+ to NMN. And the de novo NMN biosynthesis route was optimized and integrated with the NadV-mediated NMN biosynthetic pathways, leading to an intracellular concentration of 844.10 ± 17.40 µM NMN. Furthermore, the introduction of two transporters enhanced the uptake of NAM and the excretion of NMN, resulting in NMN production of 1293.73 ± 61.38 µM. Finally, by engineering an E. coli strain with optimized PRPP synthetase, we achieved the highest NMN production, reaching 3067.98 ± 27.25 µM after 24 h of fermentation at the shake flask level. In addition to constructing an efficient E. coli cell factory for NMN production, our findings provide new insights into understanding the NAD+ salvage pathway and its role in energy metabolism within E. coli.

Evaluation of the Leishmania Inositol Phosphorylceramide Synthase as a Drug Target Using a Chemical and Genetic Approach.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

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

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

Knockout of cryptochrome 1 disrupts circadian rhythm and photoperiodic diapause induction in the silkworm, Bombyx mori.

Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the involvement of clock gene cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species, remains unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on the involvement of cry1 gene in B. mori photoperiodism. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to control photoperiodic diapause induction during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction.

MiR-155 deficiency and hypoxia results in metabolism switch in the leukemic B-cells.

Hypoxia represents one of the key factors that stimulates the growth of leukemic cells in their niche. Leukemic cells in hypoxic conditions are forced to reprogram their original transcriptome, miRNome, and metabolome. How the coupling of microRNAs (miRNAs)/mRNAs helps to maintain or progress the leukemic status is still not fully described. MiRNAs regulate practically all biological processes within cells and play a crucial role in the development/progression of leukemia. In the present study, we aimed to uncover the impact of hsa-miR-155-5p (miR-155, MIR155HG) on the metabolism, proliferation, and mRNA/miRNA network of human chronic lymphocytic leukemia cells (CLL) in hypoxic conditions. As a model of CLL, we used the human MEC-1 cell line where we deleted mature miR-155 with CRISPR/Cas9. We determined that miR-155 deficiency in leukemic MEC-1 cells results in lower proliferation even in hypoxic conditions in comparison to MEC-1 control cells. Additionally, in MEC-1 miR-155 deficient cells we observed decreased number of populations of cells in S phase. The miR-155 deficiency under hypoxic conditions was accompanied by an increased apoptosis. We detected a stimulatory effect of miR-155 deficiency and hypoxia at the level of gene expression, seen in significant overexpression of EGLN1, GLUT1, GLUT3 in MEC-1 miR-155 deficient cells. MiR-155 deficiency and hypoxia resulted in increase of glucose and lactate uptake. Pyruvate, ETC and ATP were reduced. To conclude, miR-155 deficiency and hypoxia affects glucose and lactate metabolism by stimulating the expression of glucose transporters as GLUT1, GLUT3, and EGLN1 [Hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2)] genes in the MEC-1 cells.

Nonviral CRISPR/Cas9 mutagenesis for streamlined generation of mouse lung cancer models.

Functional analysis in mouse models is necessary to establish the involvement of a set of genetic variations in tumor development. A modeling platform to facilitate and cost-effectively analyze the role of multiple genes in carcinogenesis would be valuable. Here, we present an innovative strategy for lung mutagenesis using CRISPR/Cas9 ribonucleoproteins delivered via cationic polymers. This approach allows the simultaneous inactivation of multiple genes. We validate the effectiveness of this system by targeting a group of tumor suppressor genes, specifically Rb1, Rbl1, Pten, and Trp53, which were chosen for their potential to cause lung tumors, namely small cell lung carcinoma (SCLC). Tumors with histologic and transcriptomic features of human SCLC emerged after intratracheal administration of CRISPR/polymer nanoparticles. These tumors carried loss-of-function mutations in all four tumor suppressor genes at the targeted positions. These findings were reproduced in two different pure genetic backgrounds. We provide a proof of principle for simplified modeling of lung tumorigenesis to facilitate functional testing of potential cancer-related genes.

The Trypanosoma cruzi pleiotropic protein P21 orchestrates the intracellular retention and in-vivo parasitism control of virulent Y strain parasites.

P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.

Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.

L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.

Mutation-induced LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome.

Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1L580P by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1L580P-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1L580P missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.

Loss of bmp15 function in the seasonal spawner Atlantic salmon results in ovulatory failure.

Bone morphogenetic protein 15 (BMP15) is an oocyte-specific growth factor important for successful female reproduction in mammals. While mutations in BMP15/Bmp15 cause ovulatory deficiency and/or infertility in certain mammalian species, loss of bmp15 in zebrafish, a continuous spawner and the only bmp15 knockout model in fish to date, results in complete arrest of follicle development and later female-to-male sex reversal, preventing to examine effects on ovulation/fertilization. Here, we used Atlantic salmon, a seasonal spawner, and generated bmp15 mutants to investigate ovarian development and fertility. Histological and morphometric analyses revealed that in biallelic frameshift (bmp15 fs/fs) mutant ovaries, folliculogenesis started earlier, resulting in an advanced development compared to wild-type (WT) controls, accompanied by a weaker expression of the (early) oocyte-specific factor figla. This precocious ovarian development was followed in bmp15 fs/fs females by enhanced follicle atresia during vitellogenic stages. Although genes involved in steroid synthesis and signaling (star, cyp11b, cyp17a1 and esr1) were dramatically higher in late vitellogenic bmp15 fs/fs mutant ovaries, estradiol-17ß plasma levels were lower than in WT counterparts, potentially reflecting compensatory changes at the level of ovarian gene expression. At spawning, bmp15 fs/fs females displayed lower gonado-somatic index values and reduced oocyte diameter, and the majority (71.4%), showed mature non-ovulating ovaries with a high degree of atresia. The remaining (28.6%) females spawned eggs but they either could not be fertilized or, upon fertilization, showed severe malformations and embryonic mortality. Our results show that Bmp15 is required for proper follicle recruitment and growth and later ovulatory success in Atlantic salmon, providing an alternative candidate target to induce sterility in farmed salmon. Moreover, since loss of bmp15 in salmon, in contrast to zebrafish, does not result in female-to-male sex change, this is the first mutant model in fish allowing further investigations on Bmp15-mediated functions in the ovulatory period.

ATM and ATR gene editing mediated by CRISPR/Cas9 in Chinese Hamster cells.

Chinese hamster-derived cell lines including Chinese hamster lung fibroblasts (V79) have been used as model somatic cell lines in radiation biology and toxicology research for decades and have been instrumental in advancing our understanding of DNA damage response (DDR) mechanisms. Whereas many mutant lines deficient in DDR genes have been generated more than over decades, several key DDR genes such as ATM and ATR have not been established in the Chinese hamster system. Here, we transfected CRISPR/Cas9 vectors targeting Chinese hamster ATM or ATR into V79 cells and investigated whether the isolated clones had the characteristics reported in human and mouse studies. We obtained two clones of ATM knockout cells containing an insertion or deletions in the targeted locus. The ATM knockouts with no detectable ATM protein expression exhibited increased sensitivity to radiation and DNA double strand break inducing agents, cell cycle checkpoint defects and defective chromatid break repair. These are all characteristics of defective ATM function. Among the obtained ATR cells, which contained mutations in both ATR alleles while maintaining normal levels of ATR protein expression, one clone exhibited hypersensitivity to UV and replication stress agents. In the present study, we successfully established CRISPR-Cas9 derived ATM knockout cells. We couldn't knock out the ATR gene but obtained ATR mutant cells. Our results showed that Chinese hamster origin ATM knockout cells and ATR mutant cells could be useful tools for further research to reveal oncogenic functions and effects of developing anti-cancer therapeutics.

Triterpenoids-templated self-assembly nanosystem for biomimetic delivery of CRISPR/Cas9 based on the synergy of TLR-2 and ICB to enhance HCC immunotherapy.

Combination immunotherapy has shown promising potential for enhancing the objective response rate compared to immune checkpoint blockade (ICB) monotherapy. However, combination therapy with multi-drugs is limited by the different properties of the agents and inconsistent synergistic targeted delivery. Herein, based on a universal triterpene template and the anticancer active agent ursolic acid (UA), a cytomembrane-coated biomimetic delivery nanoplatform (UR@M) prepared by the self-assembly of a PD-L1 targeted CRISPR/Cas9 system and UA was designed for hepatocellular carcinoma (HCC) treatment. UR@M showed enhanced tumor accumulation in vivo with homologous tumor targeting, and CRISPR in the nanosystem exhibited potent gene-editing efficiency of 76.53% in vitro and 62.42% in vivo with no off-target effects. UA activated the natural immune system through the TLR-2-MyD88-TRAF6 pathway, which synergistically enhanced the proliferation of natural killer cells and dendritic cells and realized excellent immune cytotoxic T cell infiltration by combining with the ICB of PD-L1. The strategy of work along both lines based on innate immune and adaptive immunity displayed a significant effect in tumor regression. Overall, the UA-templated strategy "killed three birds with one stone" by establishing a self-assembly nanosystem, inducing tumor cell death, and promoting synergistic immunostimulation for HCC treatment.

OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.

Creation of an Isogenic Human iPSC-Based RGC Model of Dominant Optic Atrophy Harboring the Pathogenic Variant c.1861C>T (p.Gln621Ter) in the OPA1 Gene.

Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.