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A determining factor for insect feeding preference in the silkworm, Bombyx mori.
Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.
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Utilizing Stable Gene-Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells.
Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production. To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor-intensive process of generating and evaluating individual clones. In this study, we present a robust, high-throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late-stage viability in fed-batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5-fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.
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Stress responses. Mutations in a translation initiation factor identify the target of a memory-enhancing compound.
The integrated stress response (ISR) modulates messenger RNA translation to regulate the mammalian unfolded protein response (UPR), immunity, and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action, we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR. Clustered missense mutations were found at the amino-terminal portion of the delta subunit of guanine nucleotide exchange factor (GEF) eIF2B. When reintroduced by CRISPR-Cas9 gene editing of wild-type cells, these mutations reversed both ISRIB-mediated inhibition of the ISR and its stimulatory effect on eIF2B GEF activity toward its substrate, the translation initiation factor eIF2, in vitro. Thus, ISRIB targets an interaction between eIF2 and eIF2B that lies at the core of the ISR.
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CRISPR-cas gene-editing as plausible treatment of neuromuscular and nucleotide-repeat-expansion diseases: A systematic review.
INTRODUCTION: The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (cas) is a new technology that allows easier manipulation of the genome. Its potential to edit genes opened a new door in treatment development for incurable neurological monogenic diseases (NMGDs). The aim of this systematic review was to summarise the findings on the current development of CRISPR-cas for therapeutic purposes in the most frequent NMGDs and provide critical assessment. METHODS AND DATA ACQUISITION: We searched the MEDLINE and EMBASE databases, looking for original studies on the use of CRISPR-cas to edit pathogenic variants in models of the most frequent NMGDs, until end of 2017. We included all the studies that met the following criteria: 1. Peer-reviewed study report with explicitly described experimental designs; 2. In vitro, ex vivo, or in vivo study using human or other animal biological systems (including cells, tissues, organs, organisms); 3. focusing on CRISPR as the gene-editing method of choice; and 5. featured at least one NMGD. RESULTS: We obtained 404 papers from MEDLINE and 513 from EMBASE. After removing the duplicates, we screened 490 papers by title and abstract and assessed them for eligibility. After reading 50 full-text papers, we finally selected 42 for the review. DISCUSSION: Here we give a systematic summary on the preclinical development of CRISPR-cas for therapeutic purposes in NMGDs. Furthermore, we address the clinical interpretability of the findings, giving a comprehensive overview of the current state of the art. Duchenne's muscular dystrophy (DMD) paves the way forward, with 26 out of 42 studies reporting different strategies on DMD gene editing in different models of the disease. Most of the strategies aimed for permanent exon skipping by deletion with CRISPR-cas. Successful silencing of the mHTT gene with CRISPR-cas led to successful reversal of the neurotoxic effects in the striatum of mouse models of Huntington's disease. Many other strategies have been explored, including epigenetic regulation of gene expression, in cellular and animal models of: myotonic dystrophy, Fraxile X syndrome, ataxias, and other less frequent dystrophies. Still, before even considering the clinical application of CRISPR-cas, three major bottlenecks need to be addressed: efficacy, safety, and delivery of the systems. This requires a collaborative approach in the research community, while having ethical considerations in mind.
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TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing.
In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-β), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-β-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-β-induced EndMT. Using these techniques, we provide evidence that TGF-β2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.
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Breast Cancer Transcriptional Regulatory Network Reprogramming by using the CRISPR/Cas9 System: An Oncogenetics Perspective.
Breast cancer (BC) is the second most commonly diagnosed cancer in the world. BC develops due to dysregulation of transcriptional profiles, substantial interpatient variations, genetic mutations, and dysregulation of signaling pathways in breast cells. These events are regulated by many genes such as BRCA1/2, PTEN, TP53, mTOR, TERT, AKT, PI3K and others genes. Treatment options for BC remain a hurdle, which warrants a comprehensive understanding that establishes an interlinking connection between these genes in BC tumorigenesis. Consequently, there is an increasing demand for alternative treatment approaches and the design of more effective treatments. In this regard, it is crucial to build the corresponding transcriptional regulatory networks governing BC by using advanced genetic tools and techniques. In the past, several molecular editing technologies have been used to edit genes with several limitations. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (CRISPR/Cas9) recently received wise attention due to its potential in biomedical and therapeutic applications. Here, we review the role of various molecular signalling pathways dysregulated in BC development such as PTEN/PI3K/AKT/mTOR as well as BRCA1/BRCA2/TP53/TERT and their interplay between the related gene networks in BC initiation, progression and development of resistance against available targeted therapeutic agents. Use of CRISPR/Cas9 gene-editing technology to generate BC gene-specific transgenic cell lines and animal models to decipher their role and interactions with other gene products has been employed successfully. Moreover, the significance of using CRISPR/Cas9 technology to develop early BC diagnostic tools and treatments is discussed here.
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Human Papillomavirus Oncogene Manipulation Using Clustered Regularly Interspersed Short Palindromic Repeats/Cas9 Delivered by pH-Sensitive Cationic Liposomes.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology enables targeted gene editing, but cancer gene therapy with this approach requires improvements to enable safe and efficient delivery of CRISPR/Cas9 to tumors. We developed and evaluated a self-assembled liposome to selectively deliver CRISPR/Cas9 to cancer tissues. Our CRISPR/Cas9 system effectively inhibited proliferation of human papillomavirus (HPV) 16-positive cervical cancer cells and induced apoptosis by inactivating the HR-HPV16E6/E7 oncogene. Based on this system, we prepared a long-circulating pH-sensitive cationic nano-liposome complex with a high cell targeting and gene knockout rate. Intratumoral injection of cationic liposomes targeted to splicing HPV16 E6/E7 in nude mice significantly inhibited tumor growth without significant toxicity in vivo. Liposomes that targeted HPV16 E6/E7 splicing were established as a basis for treatment of HPV16-positive cervical cancer drug candidates. Our study demonstrates that this liposome offers an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine-based cancer therapeutics.
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Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.
The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.
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Applications of the CRISPR/Cas9 System for Rice Grain Quality Improvement: Perspectives and Opportunities.
Grain quality improvement is a key target for rice breeders, along with yield. It is a multigenic trait that is simultaneously influenced by many factors. Over the past few decades, breeding for semi-dwarf cultivars and hybrids has significantly contributed to the attainment of high yield demands but reduced grain quality, which thus needs the attention of researchers. The availability of rice genome sequences has facilitated gene discovery, targeted mutagenesis, and revealed functional aspects of rice grain quality attributes. Some success has been achieved through the application of molecular markers to understand the genetic mechanisms for better rice grain quality; however, researchers have opted for novel strategies. Genomic alteration employing genome editing technologies (GETs) like clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for reverse genetics has opened new avenues of research in the life sciences, including for rice grain quality improvement. Currently, CRISPR/Cas9 technology is widely used by researchers for genome editing to achieve the desired biological objectives, because of its simple targeting. Over the past few years many genes that are related to various aspects of rice grain quality have been successfully edited via CRISPR/Cas9 technology. Interestingly, studies on functional genomics at larger scales have become possible because of the availability of GETs. In this review, we discuss the progress made in rice by employing the CRISPR/Cas9 editing system and its eminent applications. We also elaborate possible future avenues of research with this system, and our understanding regarding the biological mechanism of rice grain quality improvement.
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A Broad Application of CRISPR Cas9 in Infectious, Inflammatory and Neurodegenerative Diseases.
Being the most important immune-responsive cell type of the CNS, microglia always glorify the so-called crossroad of Neurology, Immunology and Pharmacology. As microglial activation is a hallmark of different neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), HIV-associated neurocognitive disorders (HAND), Amyotrophic lateral sclerosis (ALS), etc., selective targeting of microglial cell signaling may be a valid option to control these neurodegenerative disorders with lesser side effects. This is particularly important as no effective therapies are available against these diseases and available neuroimmune modulators are known to target multiple cell types in a non-cell-specific manner. How we can achieve such specificity? A newly-developed cutting-edge molecular biology tool is rocking biomedical research in recent years so much so that it has already come under major lawsuits between the University of California Berkeley and the MIT-Harvard Broad Institute regarding its ownership rights, probably halting the Nobel committee to announce the most coveted prize to its owners. It is none other than Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In nutshell, the Cas9 enzyme has been paired with the bacterial immune system, CRISPR, to ultimately turn CRISPR/Cas9 as an effective genome editor. Therefore, this special issue has been devoted to highlight some of the recent discoveries on CRISPR/Cas9 in neurodegenerative disorders and explain these discoveries in the light of neuroimmune pharmacology.
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