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Editing of DNA Methylation Using dCas9-Peptide Repeat and scFv-TET1 Catalytic Domain Fusions.
DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are very important, and many epigenome editing technologies have been developed, based on zinc finger proteins, TALEs, and CRISPR/dCas9 targeting. We describe a protocol to induce and assess DNA demethylation on a target gene. It is based on a modification of the dCas9-SunTag system for efficient, targeted demethylation at specific DNA loci. The original SunTag system consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids.
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Stimuli-responsive nanoformulations for CRISPR-Cas9 genome editing.
The CRISPR-Cas9 technology has changed the landscape of genome editing and has demonstrated extraordinary potential for treating otherwise incurable diseases. Engineering strategies to enable efficient intracellular delivery of CRISPR-Cas9 components has been a central theme for broadening the impact of the CRISPR-Cas9 technology. Various non-viral delivery systems for CRISPR-Cas9 have been investigated given their favorable safety profiles over viral systems. Many recent efforts have been focused on the development of stimuli-responsive non-viral CRISPR-Cas9 delivery systems, with the goal of achieving efficient and precise genome editing. Stimuli-responsive nanoplatforms are capable of sensing and responding to particular triggers, such as innate biological cues and external stimuli, for controlled CRISPR-Cas9 genome editing. In this Review, we overview the recent advances in stimuli-responsive nanoformulations for CRISPR-Cas9 delivery, highlight the rationale of stimuli and formulation designs, and summarize their biomedical applications.
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Advances in the application of genetic manipulation methods to apicomplexan parasites.
Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterised parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods, such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation methods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of veterinary interest will ultimately lead to the development of novel and more efficient methods for disease control.
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PCdb: A comprehensive plant genome-editing database integrating sgRNA efficiency, off-target predictions, and epigenomic landscapes.
CRISPR-Cas-based genome editing has transformed plant biotechnology by enabling precise genomic modifications for crop improvement and functional genomics. The success of these applications hinges on the design of single guide RNAs (sgRNAs) that maximize on-target efficiency while minimizing off-target effects. However, existing resources for sgRNA design and performance evaluation in plants remain fragmented and lack integration with genomic and epigenomic contexts that influence both editing efficacy and specificity. Here, we present PCdb (Plant CRISPR Database; https://gmo.sjtu.edu.cn/pcdb), a comprehensive plant-focused database that integrates experimentally validated sgRNAs, annotated genomic contexts, genome-wide off-target predictions, and multilayer epigenomic annotations. PCdb encompasses 6172 manually curated editing records from 2132 publications, covering 4320 unique sgRNAs and 6 117 424 predicted off-target sites across nine major plant species. Notably, PCdb contextualizes potential editing outcomes-both on target and off target-within the chromatin landscape by incorporating DNA methylation profiles, chromatin accessibility data, and histone modification patterns. The database features an intuitive web interface that supports flexible queries, interactive visualization tools, and comprehensive analytical modules for sgRNA efficiency assessment and off-target analysis. A case study reanalysis of Oryza sativa yield-related genes demonstrates PCdb's ability to generate a detailed performance profile by evaluating both on-target characteristics and off-target risks within their native epigenomic context. Systematic analysis of the database further identifies key sequence and chromatin features that influence editing outcomes, providing novel insights to improve gene-editing efficacy and specificity.
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Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.
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CRISPR/Cas correction of muscular dystrophies.
Muscular dystrophies are a heterogeneous group of monogenic neuromuscular disorders which lead to progressive muscle loss and degeneration of the musculoskeletal system. The genetic causes of muscular dystrophies are well characterized, but no effective treatments have been developed so far. The discovery and application of the CRISPR/Cas system for genome editing offers a new path for disease treatment with the potential to permanently correct genetic mutations. The post-mitotic and multinucleated features of skeletal muscle provide an ideal target for CRISPR/Cas therapeutic genome editing because correction of a subpopulation of nuclei can provide benefit to the whole myofiber. In this review, we provide an overview of the CRISPR/Cas system and its derivatives in genome editing, proposing potential CRISPR/Cas-based therapies to correct diverse muscular dystrophies, and we discuss challenges for translating CRISPR/Cas genome editing to a viable therapy for permanent correction of muscular dystrophies.
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CRISPR Cas9 based genome editing in inherited retinal dystrophies.
BACKGROUND: Precision genome engineering, with targeted therapy towards patient-specific mutations is predicted to be the future of personalized medicine. Ophthalmology is in the frontiers of development of targeted therapy since the eye is an accessible organ and has the ease of both delivery as well as monitoring effects of therapy. MATERIALS AND METHODS: We reviewed literature using keywords CRISPR, precision medicine, genomic editing, retinal dystrophies, retinitis pigmentosa, Usher syndrome, Stargardt's Disease. Further, we collated data on current clinical trials. RESULTS: There is growing evidence on the role of genomic editing in retinal dystrophies, the various methods used, and stage of development of different therapies have been summarized in this paper. CONCLUSIONS: The CRISPR-Cas9 system has revolutionized genome editing, and opened avenues in drug discovery. It is important to understand the role of this system along with its applicability in the field of ophthalmology. In this review article, we briefly describe its methodology, the strategies of employing it for making genetic perturbations, and explore its applications in inherited retinal dystrophies.
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Genome Editing As an Approach to the Study of in Vivo Transcription Reprogramming.
CDK8-mediated transcriptional reprogramming is essential for an extensive gene expression. Constitutive knockouts of the cdk8 gene are lethal at the morula stage. For modeling transcriptional reprogramming in an adult organism, we investigated the possibility to attenuate the CDK8 kinase activity with a F97G mutation in exon 3 of the cdk8 gene. According to preliminary experimental data, this mutation should lead to a decrease in CDK8 kinase activity. To edit the genome of laboratory mice, the CRISPR/Cas9 technology was used, in which the introduction of a double-stranded gap occurred at a distance of 128 nucleotide pairs from the planned site of the introduced mutation. To introduce the mutation, a matrix for homologous repair was used as part of plasmid DNA, with homologous arms 903 and 484 bp in the 5'-3' region from the point of double-stranded rupture, respectively. As a result, mice with site-specific target mutations in exon 3 of the cdk8 gene were obtained. We for the first time demonstrated a high efficacy of the mutation 128 bp apart from the site of double-strand break. Viable animals with the F97G mutation in the catalytic domain of CDK8 kinase were obtained for the first time. The resulting cdk8 mutant mice will be used in subsequent studies to simulate the processes involving transcription reprogramming.
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CRISPR-Cas-Mediated Gene Knockout in Tomato.
Loss-of-function mutants are crucial for plant functional genomics studies. With the advent of CRISPR-Cas genome editing, generating null alleles for one or multiple specific gene(s) has become feasible for many plant species including tomato (Solanum lycopersicum). An easily programmable RNA-guided Cas endonuclease efficiently creates DNA double-strand breaks (DSBs) at targeted genomic sites that can be repaired by nonhomologous end joining (NHEJ) typically leading to small insertions or deletions that can produce null mutations. Here, we describe how to utilize CRISPR-Cas genome editing to obtain stable tomato gene knockout lines.
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Recent advances and applications of the CRISPR-Cas system in the gene therapy of blood disorders.
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