CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

The impact of Cas9-mediated mutagenesis of genes encoding potato starch-branching enzymes on starch structural properties and in vitro digestibility.

The digestibility of starch is affected by amylose content, and increasing amylopectin chain length which can be manipulated by alterations to genes encoding starch-branching enzymes (SBEs). We investigated the impact of Cas9-mediated mutagenesis of SBEs in potato on starch structural properties and digestibility. Four potato starches with edited SBE genes were tested. One lacked SBE1 and SBE2, two lacked SBE2 and had reduced SBE1, and one had reduced SBE2 only. Starch structure and thermal properties were characterised by DSC and XRD. The impact of different thermal treatments on digestibility was studied using an in vitro digestion protocol. All native potato starches were resistant to digestion, and all gelatinised starches were highly digestible. SBE modified starches had higher gelatinisation temperatures than wild type potatoes and retrograded more rapidly. Gelatinisation and 18 h of retrogradation, increased gelatinisation enthalpy, but this did not translate to differences in digestion. Following 7 days of retrogradation, starch from three modified SBE starch lines was less digestible than starch from wild-type potatoes, likely due to the recrystallisation of the long amylopectin chains. Our results indicate that reductions in SBE in potato may be beneficial to health by increasing the amount of fibre reaching the colon after retrogradation.

Leaf starch degradation by ß-amylase ZmBAM8 influences drought tolerance in maize.

As a typical C4 plant and important crop worldwide, maize is susceptible to drought. In maize, transitory starch (TS) turnover occurs in the vascular bundle sheath of leaves, differing from that in Arabidopsis (a C3 plant). This process, particularly its role in drought tolerance and the key starch-hydrolyzing enzymes involved, is not fully understood. We discovered that the expression of the ß-amylase (BAM) gene ZmBAM8 is highly upregulated in the drought-tolerant inbred line Chang7-2t. Inspired by this finding, we systematically investigated TS degradation in maize lines, including Chang7-2t, Chang7-2, B104, and ZmBAM8 overexpression (OE) and knockout (KO) lines. We found that ZmBAM8 was significantly induced in the vascular bundle sheath by drought, osmotic stress, and abscisic acid. The stress-induced gene expression and chloroplast localization of ZmBAM8 align with the tissue and subcellular sites where TS turnover occurs. The recombinant ZmBAM8 was capable of effectively hydrolyzing leaf starch. Under drought conditions, the leaf starch in ZmBAM8-OE plants substantially decreased under light, while that in ZmBAM8-KO plants did not decrease. Compared with ZmBAM8-KO plants, ZmBAM8-OE plants exhibited increased drought tolerance. Our study provides insights into the significance of leaf starch degradation in C4 crops and contributes to the development of drought-resistant maize.

Biotechnological frontiers in harnessing allelopathy for sustainable crop production.

Allelopathy, the phenomenon in which plants release biochemical compounds that influence the growth and development of neighbouring plants, presents promising opportunities for revolutionizing agriculture towards sustainability. This abstract explores the role of biotechnological advancements in unlocking the potential of allelopathy for sustainable crop production and its applications in agriculture, ecology, and natural resource management. By combining molecular, genetic, biochemical, and bioinformatic tools, researchers can unravel the complexities of allelopathic interactions and their potential for sustainable crop production and environmental stewardship. The development of novel management methods for weed control is getting a lot of attention with the introduction of new genetic technologies such as Gene drive, Transgene technologies, Gene silencing, Marker-assisted selection (MAS), and Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). By strengthening competitive characteristics these tools hold great promise for boosting crops' ability to compete with weeds. Considering recent literature, this review highlights the genetic, transcriptomics, and metabolomics approaches to allelopathy. Employing allelopathic properties in agriculture offer sustainable benefits like natural weed management, pest management, and reduced chemical pollution, but challenges include environmental factors, toxicity, regulatory hurdles, and limited resources. Effective integration requires continued research, regulatory support, and farmer education​. Also, we aimed to identify the biotechnological domains requiring more investigation and to provide the basis for future advances through this assessment.

Genome-wide CRISPR/Cas9 library screen identifies C16orf62 as a host dependency factor for porcine deltacoronavirus infection.

ABSTRACTPorcine deltacoronavirus (PDCoV) is an emerging pathogen that can cause severe diarrhea and high mortality in suckling piglets. Moreover, evidence of PDCoV infection in humans has raised concerns regarding potential public health risks. To identify potential therapeutic targets for PDCoV, we performed a genome-wide CRISPR/Cas9 library screening to find key host factors important to PDCoV infection. Several host genes in this screen were enriched, including ANPEP, which encodes the PDCoV receptor aminopeptidase N (APN). Furthermore, we discovered C16orf62, also known as the VPS35 endosomal protein sorting factor like (VPS35L), as an important host factor required for PDCoV infection. C16orf62 is an important component of the multiprotein retriever complex involved in protein recycling in the endosomal compartment and its gene knockout led to a remarkable decrease in the binding and internalization of PDCoV into host cells. While we did not find evidence for direct interaction between C16orf62 and the viral s (spike) protein, C16orf62 gene knockout was shown to downregulate APN expression at the cell surface. This study marks the first instance of a genome-wide CRISPR/Cas9-based screen tailored for PDCoV, revealing C16orf62 as a host factor required for PDCoV replication. These insights may provide promising avenues for the development of antiviral drugs against PDCoV infection.

Revolutionizing soybean genomics: How CRISPR and advanced sequencing are unlocking new potential.

Soybean Glycine max L., paleopolyploid genome, poses challenges to its genetic improvement. However, the development of reference genome assemblies and genome sequencing has completely changed the field of soybean genomics, allowing for more accurate and successful breeding techniques as well as research. During the single-cell revolution, one of the most advanced sequencing tools for examining the transcriptome landscape is single-cell RNA sequencing (scRNA-seq). Comprehensive resources for genetic improvement of soybeans may be found in the SoyBase and other genomics databases. CRISPR-Cas9 genome editing technology provides promising prospects for precise genetic modifications in soybean. This method has enhanced several soybean traits, including as yield, nutritional value, and resistance to both biotic and abiotic stresses. With base editing techniques that allow for precise DNA modifications, the use of CRISPR-Cas9 is further increased. With the availability of the reference genome for soybeans and the following assembly of wild and cultivated soybeans, significant chromosomal rearrangements and gene duplication events have been identified, offering new perspectives on the complex genomic structure of soybeans. Furthermore, major single nucleotide polymorphisms (SNPs) linked to stachyose and sucrose content have been found through genome-wide association studies (GWAS), providing important tools for enhancing soybean carbohydrate profiles. In order to open up new avenues for soybean genetic improvement, future research approaches include investigating transcriptional divergence processes, enhancing genetic resources, and incorporating CRISPR-Cas9 technologies.

Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase.

Here, we introduce 'TICIT', targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.

Advances in breeding, biotechnology, and nanotechnological approaches to combat sheath blight disease in rice.

Sheath blight, caused by the fungus Rhizoctonia solani, is a major problem that significantly impacts rice production and can lead to substantial yield losses. The disease has become increasingly problematic in recent years due to the widespread use of high-yielding semi-dwarf rice cultivars, dense planting, and heavy application of nitrogenous fertilizers. The disease has become more challenging to manage due to its diverse host range and the lack of resistant cultivars. Despite utilizing traditional methods, the problem persists without a satisfactory solution. Therefore, modern approaches, including advanced breeding, transgenic methods, genome editing using CRISPR/Cas9 technology, and nanotechnological interventions, are being explored to develop rice plants resistant to sheath blight disease. This review primarily focuses on these recent advancements in combating the sheath blight disease.

Development of a quinic acid-induced CRISPR/Cas9 genome editing system and its application for the activation of ilicicolin H biosynthesis in Trichoderma reesei.

The CRISPR/Cas9 genome editing tool has been extensively utilized in filamentous fungi, including Trichoderma reesei. However, most existing systems employ constitutive promoters for the expression of Cas9 protein within the cells or directly introduce Cas9 protein into the cells, which often leads to continuous expression of Cas9 resulting in undesired phenotypes or increased operational cost. In this study, we identified a quinic acid (QA)-induced qai5 promoter and employed it to express Cas9, thereby establishing an inducible genome editing system in T. reesei. By utilizing this system, we successfully edited the ypr1 gene and the silent gene cluster involved in ilicicolin H synthesis using donor DNA labeling 41-bp homology arm (HA), resulting in an editing efficiency ranging from 29.2 % to 46.7 %. Consequently, biosynthesis of ilicicolin H was achieved in T. reesei. To summarize, this study presents a novel form of CRISPR/Cas9 genome editing system that enables efficient and controllable genetic modifications in T. reesei.

Efficient Metabolic Pathway Modification in Various Strains of Lactic Acid Bacteria using CRISPR/Cas9 System for elevated synthesis of Antimicrobial Compounds.

Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including compounds with antimicrobial activity including bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78% than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity.

Gene and cell therapy of human genetic diseases: Recent advances and future directions.

Disruptions in normal development and the emergence of health conditions often result from the malfunction of vital genes in the human body. Decades of scientific research have focused on techniques to modify or substitute defective genes with healthy alternatives, marking a new era in disease treatment, prevention and cure. Recent strides in science and technology have reshaped our understanding of disorders, medication development and treatment recommendations, with human gene and cell therapy at the forefront of this transformative shift. Its primary objective is the modification of genes or adjustment of cell behaviour for therapeutic purposes. In this review, we focus on the latest advances in gene and cell therapy for treating human genetic diseases, with a particular emphasis on FDA and EMA-approved therapies and the evolving landscape of genome editing. We examine the current state of innovative gene editing technologies, particularly the CRISPR-Cas systems. As we explore the progress, ethical considerations and prospects of these innovations, we gain insight into their potential to revolutionize the treatment of genetic diseases, along with a discussion of the challenges associated with their regulatory pathways. This review traces the origins and evolution of these therapies, from conceptual ideas to practical clinical applications, marking a significant milestone in the field of medical science.

Genome editing of DWARF and SELF-PRUNING rapidly confers traits suitable for plant factories while retaining useful traits in tomato.

Plant factories with artificial light are less affected than open-air areas to environmental factors in crop cultivation and are attracting attention as one of the solutions to the world's food problems. However, the cost of cultivation in plant factories is higher than open-air cultivation, and currently, profitable factory-grown crop varieties are limited to those that are small or have a short growing period. Tomatoes are one of the main crops consumed around the world, but due to their large plant height and width, they are not yet suitable for mass production in plant factories. In this study, the DWARF (D) and SELF-PRUNING (SP) genes of the GABA hyperaccumulating tomato variety #87-17 were genome-edited by the CRISPR-Cas9 method to produce dwarf tomato plants. The desired traits were obtained in the T1 genome-edited generation, and the fruit traits were almost the same as those of the original variety. On the other hand, the F2 cross between #87-17 and Micro-Tom containing the d and sp mutations was dwarfed, but the fruit phenotype was a mixture of the traits of the two varieties. This indicates that genome editing of these two genes using CRISPR-Cas9 can efficiently impart traits suitable for plant factory cultivation while retaining the useful traits of the original cultivar.

Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

An evaluation of exagamglogene autotemcel for the treatment of sickle cell disease and transfusion-dependent beta-thalassaemia.

INTRODUCTION: Sickle cell disease is the most common hereditary hemoglobinopathy followed by beta-thalassemia. Until recently, allogeneic stem cell transplantation was the only curative approach. Based on the Crispr-Cas9-technology enabling targeting specific genes of interest, fetal hemoglobin which is normally shut-off after birth can be switched on and sufficient levels can alleviate symptoms in sickle cell disease and avoid transfusions in beta-thalassemia. Two first-in-human clinical studies in sickle cell disease and beta-thalassemia aiming to increase the level of fetal hemoglobin by using Crispr-Cas9 to modify autologous hematopoietic stem cells in patients aged 12-35 years have proved safety and efficacy and have shown promising clinical outcomes. AREAS COVERED: The paper summarizes the outcome of the results of the two recently published clinical studies and compares them with the other available curative approaches. EXPERT OPINION: Based on the currently available safety and efficacy data of the two published clinical results on gene therapy with Crispr-Cas9 modified autologous stem cells (exagamglogene autotemcel), it can be anticipated that this approach will add significantly to the therapeutic options for patients with sickle cell disease and beta-thalassemia and can be considered for all patients above 12 years of age independent of a suitable allogeneic stem cell donor.

CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins, paving the way for gliadin-free wheat.

Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this work, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger or NGS sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by RP-HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.

Nanoneedle Array-Electroporation Facilitates Intranuclear Ribonucleoprotein Delivery and High Throughput Gene Editing.

Dendritic cells (DCs) are critical regulators of T cell immunity, with immense therapeutic potential against tumors and autoimmune diseases. Efficient gene editing in DCs is crucial for understanding their regulatory mechanisms and maximizing their therapeutic efficacy. However, DCs are notoriously difficult to transfect, posing a major bottleneck for conventional DNA and RNA-based editing approaches. Microneedle-mediated injection of Cas9/sgRNA ribonucleoprotein (RNP) directly into the nucleus, akin to gene editing in reproductive cells, offers promise but suffers from limitations in scalability. Here, an intranuclear delivery system using a hollow nanoneedle array (HNA) combined with nano-electroporation is developed. The 2 µm-high HNA physically reaches the nucleus, positioning the nuclear envelope and plasma membrane in close proximity at the tip. Transient electronic pulses then induce simultaneous perforations across all 3 membranes, enabling direct RNP delivery into the nucleus. This HNA-based system achieves efficient knockout of genes like PD-L1 in primary DCs, demonstrating its potential as a powerful tool for gene editing in DCs and other hard-to-transfect cells.

Gene therapy for polygenic or complex diseases.

Gene therapy utilizes nucleic acid drugs to treat diseases, encompassing gene supplementation, gene replacement, gene silencing, and gene editing. It represents a distinct therapeutic approach from traditional medications and introduces novel strategies for genetic disorders. Over the past two decades, significant advancements have been made in the field of gene therapy, leading to the approval of various gene therapy drugs. Gene therapy was initially employed for treating genetic diseases and cancers, particularly monogenic conditions classified as orphan diseases due to their low prevalence rates; however, polygenic or complex diseases exhibit higher incidence rates within populations. Extensive research on the etiology of polygenic diseases has unveiled new therapeutic targets that offer fresh opportunities for their treatment. Building upon the progress achieved in gene therapy for monogenic diseases and cancers, extending its application to polygenic or complex diseases would enable targeting a broader range of patient populations. This review aims to discuss the strategies of gene therapy, methods of gene editing (mainly CRISPR-CAS9), and carriers utilized in gene therapy, and highlight the applications of gene therapy in polygenic or complex diseases focused on applications that have either entered clinical stages or are currently undergoing clinical trials.

Phenazines are involved in the antagonism of a novel subspecies of Pseudomonas chlororaphis strain S1Bt23 against Pythium ultimum.

Long-term use of chemical fungicides to control plant diseases caused by fungi and oomycetes has led to pathogen resistance and negative impacts on public health and environment. There is a global search for eco-friendly methods and antagonistic bacteria are emerging as alternatives. We isolated a potent antagonistic bacterial strain (S1Bt23) from woodland soil in Québec, Canada. Taxonomic characterization by 16S rRNA, multi-locus sequence analysis, pairwise whole-genome comparisons, phylogenomics and phenotypic data identified strain S1Bt23 as a novel subspecies within Pseudomonas chlororaphis. In dual culture studies, strain S1Bt23 exhibited potent mycelial growth inhibition (60.2-66.7%) against Pythium ultimum. Furthermore, strain S1Bt23 was able to significantly bioprotect potato tuber slices from the development of necrosis inducible by P. ultimum. Annotations of the whole genome sequence of S1Bt23 revealed the presence of an arsenal of secondary metabolites including the complete phenazine biosynthetic cluster (phzABCDEFG). Thin-layer (TLC) and high-performance liquid (HPLC) chromatographic analyses of S1Bt23 extracts confirmed the production of phenazines, potent antifungal compounds. CRISPR/Cas9-mediated deletion of phzB (S1Bt23ΔphzB) or phzF (S1Bt23ΔphzF) gene abrogated phenazine production based on TLC and HPLC analyses. Also, S1Bt23ΔphzB and S1Bt23ΔphzF mutants lost antagonistic activity and bioprotection ability of potato tubers against P. ultimum. This demonstrated that phenazines are involved in the antagonistic activity of S1Bt23 against P. ultimum. Finally, based on genotypic and phenotypic data, we taxonomically conclude that S1Bt23 represents a novel subspecies for which the name Pseudomonas chlororaphis subsp. phenazini is proposed.

Exploration of the pneumocandin biosynthetic gene cluster based on efficient CRISPR/Cas9 gene editing strategy in Glarea lozoyensis.

Pneumocandin B0 (PB0) is a lipopeptide produced by the fungus Glarea lozoyensis. The existing challenges with the low-yield and the extended-fermentation cycle emphasize necessity for strain improvement. In this study, we optimized conditions to obtain high-quality protoplasts and screened effective selection markers, leading to the construction of three CRISPR/Cas9 gene editing systems. Utilizing a constitutive Cas9 expression recipient strain, combined with dual sgRNAs targeting, we achieved highly efficient editing of target genes. We successfully knocked out 10 genes within the pneumocandin putative biosynthetic gene cluster and analyzed their roles in PB0 production. Our findings reveal that 4 of 10 genes are directly involved in PB0 production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB0 production, while the absence of glhyp or glhtyC led to the complete loss of PB0 biosynthesis. Notably, the deletion of glhyp caused the silencing of nearly all cluster genes, whereas overexpression of glhyp led to a 2.38-fold increase in PB0 production. Therefore, this study provides the first comprehensive exploration of the functions of 10 genes within the pneumocandin putative biosynthetic gene cluster. Our findings provide valuable technical strategies for constructing bioengineering strains with purposefully enhanced PB0 production.