Genome Editing Techniques Research Articles

Application of CRISPR/Cas9-mediated gene editing for abiotic stress management in crop plants.

Abiotic stresses, including drought, salinity, cold, heat, and heavy metals, extensively reducing global agricultural production. Traditional breeding approaches and transgenic technology have been widely used to mitigate the risks of these environmental stresses. The discovery of engineered nucleases as genetic scissors to carry out precise manipulation in crop stress-responsive genes and associated molecular network has paved the way for sustainable management of abiotic stress conditions. In this context, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas)-based gene-editing tool has revolutionized due to its simplicity, accessibility, adaptability, flexibility, and wide applicability. This system has great potential to build up crop varieties with enhanced tolerance against abiotic stresses. In this review, we summarize the latest findings on understanding the mechanism of abiotic stress response in plants and the application of CRISPR/Cas-mediated gene-editing system towards enhanced tolerance to a multitude of stresses including drought, salinity, cold, heat, and heavy metals. We provide mechanistic insights on the CRISPR/Cas9-based genome editing technology. We also discuss applications of evolving genome editing techniques such as prime editing and base editing, mutant library production, transgene free and multiplexing to rapidly deliver modern crop cultivars adapted to abiotic stress conditions.

Abstract LB071: A phenomics platform combining imaging and artificial intelligence for rapid validation and advancement of novel oncology targets

Abstract The emergence of technological innovations has created the opportunity to envision new approaches to discover therapeutics at scale. We combined advances in high content microscopy with arrayed CRISPR genome editing techniques and machine learning (ML) to build a rigorously controlled dataset enabling exploration of biology and chemistry at scale. Phenotypes from millions of perturbations in multiple cell types were embedded in a unified representation space and leveraged to accelerate discovery and reverse translation, ultimately yielding novel biological insights, and optimizing the advancement of lead molecular series through structure-activity relationships (SAR). Here, we demonstrate the capability of our platform to discover potential cancer therapies with distinct mechanisms of action. First, we describe the identification of a novel compound series that potentiates the effects of immunotherapy in syngeneic mouse models, producing complete responses and immunological memory, while also limiting peripheral inflammation. Specific novel chemical entities (NCEs) caused robust CD45+ cell influx into the tumor microenvironment and significantly attenuated exhausted T cells and immunosuppressive macrophages, thereby enhancing anti-tumor immunity. Strikingly, the same NCEs suppressed peripheral inflammation while sustaining elevated levels of intra-tumoral proinflammatory cytokines. Second, we highlight a novel and differentiated strategy to potentiate PARP inhibitor response in homologous repair deficient (HRD) - negative or HR-proficient ovarian cancers. NCEs altered the expression of genes within the DNA damage repair (DDR) network and cell cycle checkpoints to synergize with PARP inhibition in vivo and re-sensitized a PARP-resistant patient-derived xenograft (PDX) model. Collectively, we believe future efforts on the industrialization and integration of various technological innovations across biology, chemistry, automation, data science, and engineering will ultimately modernize drug discovery and radically improve patient lives. Citation Format: Jenny Rudnick, Kiran Nadella, Chase Neumann, Shane Rowley, Ethan Gardner, Shadi Swaidani, Aimee Iberg, Lu Chen, Daria Beshnova, Aurora Blucher, Rebecca Sarto Basso, Malini Rajan, Kevin Fales, Ashraf Saeed, Christopher Bailey, Weston Judd, Chrissy Egbert, Joel Ellis, John Ansede, Pouya Hadipour, Kevin Jessing, Janet Paulsen, Paul Rearden, Vamshi Manda, Sashi Kasimsetty, Sashi Kasimsetty, Michael Hancock, Harish Shankaran, Bryan Ellis, Meenakshy Iyer, Carl Brooks, Ashish Bhandari, Chris Gibson, Irit Rappley, Laura Schaevitz, Imran Haque, Hayley Donnella, Michael Cuccarese, Marie Evangelista. A phenomics platform combining imaging and artificial intelligence for rapid validation and advancement of novel oncology targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB071.

An overview of genome engineering in plants, including its scope, technologies, progress and grand challenges.

Genome editing is a useful, adaptable, and favored technique for both functional genomics and crop enhancement. Over the years, rapidly evolving genome editing technologies, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas), transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), have shown broad application prospects in gene function research and improvement of critical agronomic traits in many crops. These technologies have also opened up opportunities for plant breeding. These techniques provide excellent chances for the quick modification of crops and the advancement of plant science in the future. The current review describes various genome editing techniques and how they function, particularly CRISPR/Cas9 systems, which can contribute significantly to the most accurate characterization of genomic rearrangement and plant gene functions as well as the enhancement of critical traits in field crops. To accelerate the use of gene-editing technologies for crop enhancement, the speed editing strategy of gene-family members was designed. As it permits genome editing in numerous biological systems, the CRISPR technology provides a valuable edge in this regard that particularly captures the attention of scientists.

<i>In vitro</i> regeneration of grape

Considering the global and Russian experience in grape accessions preservation, one of the most reliable ways is the creation of a duplicate in vitro collection. However, in connection with the creation of duplicate grape collections and development of genome editing techniques, there is a need for selecting the most optimal medium composition that will ensure the maximum rate of callus formation and regeneration during the introduction of grape plants into in vitro culture. This will make it possible to obtain the necessary amount of material for further editing and subsequent regeneration of plants with knockout of target genes to improve economically valuable traits. For grapes, this is primarily an increase in resistance to powdery mildew caused by Uncinula necator Burill.The effect of active substances of biological and synthetic origin on the morphogenesis has been studied for many crops, including regenerant plants of the genus Vitis L. However, the genus Vitis is very diverse and heterogeneous in its genetic, physiological and morphological structure, as a result of which the recommended media and components for cultivation under in vitro conditions may not suit every cultivar. Local Russian grape cultivars are better suited to local growing conditions, so efforts should be focused on the development of techniques related to the preservation of local varieties in collections in vitro.Knowledge of genes controlling certain traits, as well as the availability of grape accessions whose genome has been sequenced, contribute to successful in silico analysis for creating editing constructs.

Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome

The application of the CRISPR/Cas9-based genome editing technique to the yeast Saccharomyces cerevisiae has made it possible to simultaneously modify several sites, particularly to integrate several expression cassettes. The existing methods provide high efficiency for such modifications; however, common protocols include several preparatory steps, namely, the construction of an intermediate Cas9-expressing strain, the assembly of a plasmid bearing several single guide RNA (sgRNA) expression cassettes, and the surrounding integrated DNA fragments with long flanks for recombination with target loci. Since these preparatory steps are time consuming and may not be desirable in some types of experiments, we explored the possibility of multiple integration without these steps. We have demonstrated that it is possible to skip them simultaneously and integrate up to three expression cassettes into separate sites by transforming the recipient strain with the Cas9 expression plasmid, three differently marked sgRNA plasmids, and three donor DNAs flanked with short (70 bp) arms for recombination. This finding increases the flexibility of choosing the optimal experimental design for multiple editing of the genome of S. cerevisiae and can significantly accelerate such experiments.

Transgene-Free Genome Editing for Biotic and Abiotic Stress Resistance in Sugarcane: Prospects and Challenges

Sugarcane (Saccharum spp.) is one of the most valuable food and industrial crops. Its production is constrained due to major biotic (fungi, bacteria, viruses and insect pests) and abiotic (drought, salt, cold/heat, water logging and heavy metals) stresses. The ever-increasing demand for sugar and biofuel and the rise of new pest and disease variants call for the use of innovative technologies to speed up the sugarcane genetic improvement process. Developing new cultivars through conventional breeding techniques requires much time and resources. The advent of CRISPR/Cas genome editing technology enables the creation of new cultivars with improved resistance/tolerance to various biotic and abiotic stresses. The presence of genome editing cassette inside the genome of genome-edited plants hinders commercial exploitation due to regulatory issues. However, this limitation can be overcome by using transgene-free genome editing techniques. Transgene-free genome editing approaches, such as delivery of the RNPs through biolistics or protoplast fusion, virus-induced genome editing (VIGE), transient expression of CRISPR/Cas reagents through Agrobacterium-mediated transformation and other approaches, are discussed. A well-established PCR-based assay and advanced screening systems such as visual marker system and Transgene killer CRISPR system (TKC) rapidly identify transgene-free genome edits. These advancements in CRISPR/Cas technology speed up the creation of genome-edited climate-smart cultivars that combat various biotic and abiotic stresses and produce good yields under ever-changing conditions.

A review of synthetic biology tools in Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional oleaginous yeast with great potential for industrial production. Y. lipolytica has a high propensity for flux through tricarboxylic acid cycle intermediates. Therefore, this host is currently being developed as a workhorse, and is rapidly emerging in biotechnology fields, especially for industrial chemical production, whole-cell bioconversion, and the treatment and recycling of industrial waste. In recent studies, Y. lipolytica has been rewritten and introduced with non-native metabolites of certain compounds of interest owing to the advancement in synthetic biology tools. In this review, we collate recent progress to present a detailed and insightful summary of the major developments in synthetic biology tools and techniques for Y. lipolytica, including promoters, terminators, selection markers, autonomously replicating sequences, DNA assembly techniques, genome editing techniques, and subcellular organelle engineering. This comprehensive overview would be a useful resource for future genetic engineering studies to improve the yield of desired metabolic products in Y. lipolytica.

Restructuring plant types for developing tailor-made crops.

Plants have adapted to different environmental niches by fine-tuning the developmental factors working together to regulate traits. Variations in the developmental factors result in a wide range of quantitative variations in these traits that helped plants survive better. The major developmental pathways affecting plant architecture are also under the control of such pathways. Most notable are the CLAVATA-WUSCHEL pathway regulating shoot apical meristem fate, GID1-DELLA module influencing plant height and tillering, LAZY1-TAC1 module controlling branch/tiller angle and the TFL1-FT determining the floral fate in plants. Allelic variants of these key regulators selected during domestication shaped the crops the way we know them today. There is immense yield potential in the 'ideal plant architecture' of a crop. With the available genome-editing techniques, possibilities are not restricted to naturally occurring variations. Using a transient reprogramming system, one can screen the effect of several developmental gene expressions in novel ecosystems to identify the best targets. We can use the plant's fine-tuning mechanism for customizing crops to specific environments. The process of crop domestication can be accelerated with a proper understanding of these developmental pathways. It is time to step forward towards the next-generation molecular breeding for restructuring plant types in crops ensuring yield stability.

An Optimized Protocol for In Vitro Regeneration of Ocimum basilicum cv. FT Italiko

Sweet basil (Ocimum basilicum L.; Fam. Lamiaceae) is an annual herbaceous plant with a high economic value used in folk medicine, pharmacology, and food production. In Italy, most of the varieties are used to produce the famous “pesto” sauce; however, almost all of them are susceptible to basil downy mildew (BDM) disease, strongly decreasing the growth of the fresh leaves and the survival of the whole plant. Nowadays, CRISPR/Cas9 technology is recognized to be a prominent way to enhance basil genetic breeding. In this work, we present an optimized protocol for in vitro direct regeneration of an elite cultivar, which is the major limiting factor for the transformation of O. basilicum. Regeneration has been obtained from different explants (leaves, cotyledons, cotyledonary nodes); the highest frequency has been obtained from cotyledonary nodes of seedlings germinated on MS medium containing TDZ. This protocol may be used for biotechnological applications as genome editing techniques to obtain basil-downy-mildew-disease-resistant clones.

Parthenocarpy-related genes induced by naphthalene acetic acid in oil palm interspecific O × G [Elaeis oleifera (Kunth) Cortés × Elaeis guineensis Jacq.] hybrids.

Parthenocarpy is the development without fertilization of seedless fruits. In the oil palm industry, the development of parthenocarpic fruits is considered an attractive option to increase palm oil production. Previous studies have shown the application of synthetic auxins in Elaeis guineensis, and interspecific O×G hybrids (Elaeis oleifera (Kunth) Cortés × E. guineensis Jacq.) induces parthenocarpy. The aim of this study was to identify the molecular mechanism through transcriptomics and biology system approach to responding to how the application of NAA induces parthenocarpic fruits in oil palm O×G hybrids. The transcriptome changes were studied in three phenological stages (PS) of the inflorescences: i) PS 603, pre-anthesis III, ii) PS 607, anthesis, and iii) PS 700, fertilized female flower. Each PS was treated with NAA, Pollen, and control (any application). The expression profile was studied at three separate times: five minutes (T0), 24 hours (T1), and 48h post-treatment (T2). The RNA sequencing (RNA seq) approach was used with 27 oil palm O×G hybrids for a total of 81 raw samples. RNA-Seq showed around 445,920 genes. Numerous differentially expressed genes (DEGs) were involved in pollination, flowering, seed development, hormone biosynthesis, and signal transduction. The expression of the most relevant transcription factors (TF) families was variable and dependent on the stage and time post-treatment. In general, NAA treatment expressed differentially more genes than Pollen. Indeed, the gene co-expression network of Pollen was built with fewer nodes than the NAA treatment. The transcriptional profiles of Auxin-responsive protein and Gibberellin-regulated genes involved in parthenocarpy phenomena agreed with those previously reported in other species. The expression of 13 DEGs was validated by RT-qPCR analysis. This detailed knowledge about the molecular mechanisms involved in parthenocarpy could be used to facilitate the future development of genome editing techniques that enable the production of parthenocarpic O×G hybrid cultivars without growth regulator application.

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.

Eye on genome editing.

CRISPR/Cas9 genome editing techniques have the potential to treat previously untreatable inherited genetic disorders of vision by correcting mutations that cause these afflictions. Using a prime editor, Qin et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20220776) restored visual functions in a mouse model (rd10) of retinitis pigmentosa.

CRISPR and gene editing in HIV therapy: A scoping review

Human immunodeficiency virus (HIV) is nowadays one of the major health problems worldwide. Several strategies have been explored for the eradication of latent HIV-1 reservoirs, current antiretroviral therapy stops the progression of HIV disease, however, as new therapies are being developed, CRISPR (clustered regularly interspaced short palindromic repeats) systems have recently been used as a genome editing technique. CRISPR is becoming progressively one of the possible cures, although it remains experimental, for HIV in medical research. This systematic review summarizes literature evidence of CRISPR gene editing in HIV/AIDS therapy. This study also identifies the research gaps in the current literature, helping to guide future research.

Mapping the SLP76 interactome in T cells lacking each of the GRB2-family adaptors reveals molecular plasticity of the TCR signaling pathway.

The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations.

Plant breeding advancements with "CRISPR-Cas" genome editing technologies will assist future food security.

Genome editing techniques are being used to modify plant breeding, which might increase food production sustainably by 2050. A product made feasible by genome editing is becoming better known, because of looser regulation and widespread acceptance. The world's population and food supply would never have increased proportionally under current farming practices. The development of plants and food production has been greatly impacted by global warming and climate change. Therefore, minimizing these effects is crucial for agricultural production that is sustainable. Crops are becoming more resilient to abiotic stress because of sophisticated agricultural practices and a better understanding of the abiotic stress response mechanism. Both conventional and molecular breeding techniques have been used to create viable crop types both processes are time-consuming. Recently, plant breeders have shown an interest in genome editing approaches for genetic manipulation that use clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). To ensure the security of the food supply in the future, plant kinds with desired traits must be developed. A completely new era in plant breeding has begun because of the revolution in genome editing techniques based on the CRISPR/CRISPR-associated nuclease (Cas9) systems. All plants may effectively target a particular gene or group of loci using Cas9 and single-guide RNA (sgRNA). CRISPR/Cas9 can thereby save time and labor compared to conventional breeding methods. An easy, quick, and efficient method for directly altering the genetic sequences in cells is with the CRISPR and Cas9 systems. The CRISPR-Cas9 system, which was developed from components of the earliest known bacterial immune system, allows for targeted gene breakage and gene editing in a variety of cells/RNA sequences to guide endonuclease cleavage specificity in the CRISPR-Cas9 system. Editing can be directed to practically any genomic site by altering the guide RNA (gRNA) sequence and delivering it to a target cell along with the Cas9 endonuclease. We summarize recent CRISPR/Cas9 plant research findings, investigate potential applications in plant breeding, and make predictions about likely future breakthroughs and approaches to food security through 2050.

The multi-BRCT domain protein DDRM2 promotes the recruitment of RAD51 to DNA damage sites to facilitate homologous recombination.

DNA double-strand breaks (DSBs) are the most toxic form of DNA damage in cells. Homologous recombination (HR) is an error-free repair mechanism for DSBs as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. Through genetic screens for DNA damage response mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including bryophytes, the earliest land plant lineage. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. In support, genetic analysis suggests that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Mechanistically, DDRM2 interacts with the core HR protein RAD51 and is required for the recruitment of RAD51 to DSB sites. Our study reveals that SOG1-DDRM2-RAD51 is a novel module for HR, providing a potential target for improving the efficiency of gene targeting.

CRISPR-Cas9 as a Potential Cancer Therapy Agent: An Update

Cancer is the second leading cause of death globally and remains a major economic and social burden. Although our understanding of cancer at the molecular level continues to improve, more effort is needed to develop new therapeutic tools and approaches exploiting these advances. Due to its high efficiency and accuracy, the CRISPR-Cas9 genome editing technique has recently emerged as a cancer treatment strategy. Among its many applications, CRISPR-Cas9 has shown an unprecedented clinical potential to discover novel targets for cancer therapy and to dissect chemical-genetic interactions, providing insight into how tumors respond to drug treatment. Moreover, CRISPR-Cas9 can be employed to rapidly engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. More importantly, the ability of CRISPR-Cas9 to accurately edit genes, not only in cell culture models and model organisms but also in humans, allows its use in therapeutic explorations this review, important considerations for the use of CRISPR/Cas9 in therapeutic properties are discussed, along with major challenges that will need to be addressed before clinical examinations for a complex and polygenic disease such as cancer. This review aimed to explore the potential of the CRISPR-Cas9 genome editing technique as a cancer treatment strategy. Specifically, we will discuss how CRISPR-Cas9 can be used to discover novel targets for cancer therapy and to dissect chemical-genetic interactions.

Are CRISPR/Cas genome editing techniques the future of plant breeding?

Are CRISPR/Cas genome editing techniques the future of plant breeding?

Cardiac Xenotransplantation: a New Frontier for Advanced Heart Failure.

Cardiac transplantation is a critical treatment for patients with advanced heart failure, offering the ability to markedly improve quality and quantity of life. Unfortunately, this treatment is limited by donor organ availability, despite efforts to increase organ supply and improve donor organ allocation and usage. The transplantation of non-human donor organs (xenotransplantation) offers to readily address many limitations of the current transplantation system; however, scattered attempts to establish this practice have been met with frustration. In this review, we discuss the limitations of the historical attempts and outline recent progress in the field of cardiac xenotransplantation. The advent of CRISPR-Cas9 genome editing techniques and emerging commercial and regulatory alignment has led to a flurry of new attempts to establish xenotransplantation as a viable treatment for those with end-stage heart failure. The first xenotransplantation of a genetically modified pig heart to a human recipient on January 7, 2022, highlighted the progress the science of xenotransplantation has made, as well as the need to outline next steps to further establish the practice. The development of a genetically modified porcine model has renewed hope that xenotransplantation might succeed where prior attempts failed, though many barriers remain.

Current trends in management of bacterial pathogens infecting plants.

Plants are continuously challenged by different pathogenic microbes that reduce the quality and quantity of produce and therefore pose a serious threat to food security. Among them bacterial pathogens are known to cause disease outbreaks with devastating economic losses in temperate, tropical and subtropical regions throughout the world. Bacteria are structurally simple prokaryotic microorganisms and are diverse from a metabolic standpoint. Bacterial infection process mainly involves successful attachment or penetration by using extracellular enzymes, type secretion systems, toxins, growth regulators and by exploiting different molecules that modulate plant defence resulting in successful colonization. Theses bacterial pathogens are extremely difficult to control as they develop resistance to antibiotics. Therefore, attempts are made to search for innovative methods of disease management by the targeting bacterial virulence and manipulating the genes in host plants by exploiting genome editing methods. Here, we review the recent developments in bacterial disease management including the bioactive antimicrobial compounds, bacteriophage therapy, quorum-quenchingmediated control, nanoparticles and CRISPR/Cas based genome editing techniques for bacterial disease management. Future research should focus on implementation of smart delivery systems and consumer acceptance of these innovative methods for sustainable disease management.