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BATCH-GE: Analysis of NGS Data for Genome Editing Assessment.
Due to its simple nature, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technique is massively used nowadays to modify genomic loci in a wide range of model systems. The possibility to interrogate gene function on a genome-wide scale is revolutionizing fundamental life sciences and will lead to new clinical breakthroughs. Its strength is even more pronounced when it is used in tandem with next-generation sequencing (NGS). The high throughput and low cost cause NGS to be the method of choice for exploring CRISPR-Cas9 experimental results. To analyze the NGS reads from genome editing experiments only few bioinformatics tools are available. BATCH-GE is a flexible and easy-to-use tool, which is especially useful for dealing with large amounts of data. It detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel.
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Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.
Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.
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A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.
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Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.
Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.
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CRISPR-Cas9 based plant genome editing: Significance, opportunities and recent advances.
Precise genome editing is a quantum leap in the field of plant sciences. Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas9 protein have emerged as a powerful tool for precise genome editing. CRISPR-Cas9 system introduces small heritable mutations (indels) in the genome of an organism. This system also enables precise gene characterization in plants with complex genomes. Besides, it offers new opportunities of trait stacking, where addition of desirable traits or removal of undesirable traits can be achieved simultaneously in a single event. With CRISPR-Cas9 RNPs technology, raising transgene free genetically modified plants is within realm of possibility which would be helpful in addressing regulatory concerns of transgenic plants. Several new advancements have been made in this technology which has extended its applications in almost every aspect of plant science. For example, recently developed catalytically inactive dCas9 fused with transcriptional effector domains allows targeted activation or silencing of the gene of interest. Apart from this, dCas9 fused with fluorescent labels is a budding tool in chromatin imaging studies. In this review, we summarize these recent advancements in CRISPR/Cas system and methods for analyzing the induced mutations, and its implementations in crop improvement.
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Agrobacterium-Mediated Genetic Transformation and Genome Editing Using CRISPR-Cas9 Constructs in Rice.
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 has become an essential tool in every molecular plant breeding laboratory. CRISPR-Cas9 causes highly precise knock-out mutations in plants that can be exploited in crop improvement programmes. However, poor response to Agrobacterium-mediated genetic transformation in recalcitrant rice genotypes is a major limiting factor. This protocol describes a detailed procedure for genome editing with CRISPR-Cas9 in recalcitrant rice genotypes that otherwise show a poor response to tissue culture. With this method, high transformation efficiency can be achieved in relatively a short period.
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Crispr/Cas-based modeling of NF2 loss in meningioma cells.
BACKGROUND: Alterations of the neurofibromatosis type 2 gene (NF2) occur in more than fifty percent of sporadic meningiomas. Meningiomas develop frequently in the setting of the hereditary tumor syndrome NF2. Investigation of potential drug-based treatment options has been limited by the lack of appropriate in vitro and in vivo models. NEW METHODS: Using Crispr/Cas gene editing, of the malignant meningioma cell line IOMM-Lee, we generated a pair of cell clones characterized by either stable knockout of NF2 and loss of the protein product merlin or retained merlin protein (transfected control without gRNA). RESULTS: IOMM-Lee cells lacking NF2 showed reduced apoptosis and formed bigger colonies compared to control IOMM-Lee cells. Treatment of non-transfected IOMM-Lee cells with the focal adhesion kinase (FAK) inhibitor GSK2256098 resulted in reduced colony sizes. Orthotopic mouse xenografts showed the formation of convexity tumors typical for meningiomas with NF2-depleted and control cells. COMPARISON WITH EXISTING METHODS: No orthotopic meningioma models with genetically-engineered cell pairs are available so far. CONCLUSION: Our model based on Crispr/Cas-based gene editing provides paired meningioma cells suitable to study functional consequences and therapeutic accessibility of NF2/merlin loss.
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Efficient generation of hiPSC neural lineage specific knockin reporters using the CRISPR/Cas9 and Cas9 double nickase system.
Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.
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Genome editing in the mammalian brain using the CRISPR-Cas system.
Recent advances in genome editing technologies such as the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 have enabled the rapid and efficient modification of endogenous genomes in a variety of cell types, accelerating biomedical research. In particular, precise genome editing in somatic cells in vivo allows for the rapid generation of genetically modified cells in living animals and holds great promise for the possibility of directly correcting genetic defects associated with human diseases. However, because of the limited efficiency and suitability of these technologies in the brain, especially in postmitotic neurons, the practical application of genome editing technologies has been largely limited in the field of neuroscience. Recent technological advances overcome significant challenges facing genome editing in the brain and have enabled us to precisely edit the genome in both mitotic cells and mature postmitotic neurons in vitro and in vivo, providing powerful means for studying gene function and dysfunction in the brain. This review highlights the development of genome editing technologies for the brain and discusses their applications, limitations, and future challenges.
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Legal issues regarding gene editing at the beginning of life: an EU perspective.
The development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-modification technologies has opened impressive possibilities for the biomedical sciences. However, their application to human embryos and early fetuses has raised huge ethical and legal discussions because it affects the human germline. This paper provides a critical and in-depth analysis of the current legal framework on this topic in the EU context and at the national level in the member states. It also offers an alternative interpretation of the regulation, so as to help researchers, practitioners, policy makers and society as a whole to find efficient responses to challenges that cannot wait for a legally updated answer. As a final result, this paper will show that eugenic uses of CRISP-Cas and any kind of modification intended to alter the human germ line are generally banned in the EU context, while basic research on human embryos is mostly permitted. The legal status of therapeutic applications of CRISPR-Cas on early fetuses, however, has not been adequately addressed by the EU zone regulation.
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