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Epigenome editing technologies for discovery and medicine.
Epigenome editing has rapidly evolved in recent years, with diverse applications that include elucidating gene regulation mechanisms, annotating coding and noncoding genome functions and programming cell state and lineage specification. Importantly, given the ubiquitous role of epigenetics in complex phenotypes, epigenome editing has unique potential to impact a broad spectrum of diseases. By leveraging powerful DNA-targeting technologies, such as CRISPR, epigenome editing exploits the heritable and reversible mechanisms of epigenetics to alter gene expression without introducing DNA breaks, inducing DNA damage or relying on DNA repair pathways.
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Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.
BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.
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CRISPR/Cas system: An emerging technology in stem cell research.
The identification of new and even more precise technologies for modifying and manipulating the genome has been a challenge since the discovery of the DNA double helix. The ability to modify selectively specific genes provides a powerful tool for characterizing gene functions, performing gene therapy, correcting specific genetic mutations, eradicating diseases, engineering cells and organisms to achieve new and different functions and obtaining transgenic animals as models for studying specific diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has recently revolutionized genome engineering. The application of this new technology to stem cell research allows disease models to be developed to explore new therapeutic tools. The possibility of translating new systems of molecular knowledge to clinical research is particularly appealing for addressing degenerative diseases. In this review, we describe several applications of CRISPR/Cas9 to stem cells related to degenerative diseases. In addition, we address the challenges and future perspectives regarding the use of CRISPR/Cas9 as an important technology in the medical sciences.
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Transforming tomatoes into GABA-rich functional foods through genome editing: A modern biotechnological approach.
Gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter which blocks the impulses between nerve cells in the brain. Due to the increasing awareness about the health promoting benefits associated with GABA, it is also artificially synthesized and consumed as a nutritional supplement by people in some regions of the world. Though among the fresh vegetables, tomato fruits do contain a comparatively higher amount of GABA (0.07 to 2.01 mg g-1 FW), it needs to be further enhanced to fully impart its potential health benefits. Achieving this feat through classical breeding approaches is time and resource consuming, and is also associated with linkage drag. On the other hand, precise targeting of specific sites in the genome with less off- target effects is mediated by CRISPR/Cas9 genome editing tool and is widely used to overcome the barriers associated with traditional breeding approaches. Combining genome editing with speed breeding techniques can enable the rapid development of GABA-rich tomato cultivars, paving a way to unlock a new era of functional foods, where every bite contributes to cognitive well-being and holistic health. This review highlights the significance of GABA boosted functional foods and explores the potential of CRISPR/Cas9 technology for developing GABA enriched tomatoes.
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[Research progress in the third-generation genomic editing technology - CRISPR/Cas9].
CRISPR/Cas9 technology originated from type II CRISPR/Cas system, which is widely found in bacteria and equips them with acquired immunity against viruses and plasmids. CRISPR-associated protein Cas9 is a RNA-guided endonuclease, which can efficiently introduce double-strand breaks at specific sites and activate homologous recombination and/or non-homologous end joining mechanism for the repair of impaired DNA. Features such as easy-to-use, cost-effectiveness, multiple targeting ability have made it the third-generation genomic engineering tool following ZFNs and TALENs. Here the history of discovery and molecular mechanism of the CRISPR/Cas9 technology are reviewed. The rapid advance in its various applications, especially for the treatment of human genetic disorders, as well as some concomitant problems are discussed.
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Genome editing of polyploid crops: prospects, achievements and bottlenecks.
Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.
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Utilizing CRISPR/Cas9 technology to prepare lymphoblastoid cell lines harboring genetic mutations for generating quality control materials in genetic testing.
BACKGROUND: To meet the requirements of the rapidly progressing genetic testing technologies in clinical laboratories, assuring the quality of genetic tests by utilizing appropriate quality control materials is of paramount importance. The CRISPR/Cas9 technology was used to prepare quality control materials because genome-edited human cell lines are one of the major resources for quality control materials. METHODS: In this study, in vitro transcribed sgRNA were transfected into a Cas9-expressing lymphoblastoid cell line (LCL)-by electroporation-to simulate the SEA-type deletion observed in α-thalassemia. The edited positive cell line was screened and identified by polymerase chain reaction (PCR) followed by Sanger sequencing. The whole-genome sequencing was also performed to show evidence of predicted mutation. RESULTS: The results showed that electroporation of the in vitro transcribed gRNAs into stable Cas9-expressing LCL was a more efficient gene-editing technique as compared to plasmid-mediated transfection, and that the positive rates could reach up to 35.9%. The predominance of indel sizes relative to the predicted deletion length was clustered between 10 and 0 bp. The results of whole-genome sequencing also demonstrated the existence of SEA-type deletion of α-thalassemia. CONCLUSIONS: Gene-editing based on Cas9-expressing LCL by electroporation of sgRNA was a more efficient approach to introduce mutations for generating quality control materials for genetic testing. The edited lymphoblastoid cell lines were feasible to serve as quality control materials in genetic testing.
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Artificial Intelligence-Assisted CRISPR/Cas Systems for Targeting Plant Viruses.
Plant viral infections continue to pose a significant and ongoing threat to global food security, especially in the context of climatic instability and intensive agricultural practices. The CRISPR/Cas system has emerged as a powerful tool for developing virus-resistant crops by enabling precise modifications to viral genomes or plant susceptibility factors. Nonetheless, the efficacy and dependability of CRISPR-based antiviral approaches are limited by challenges in guide RNA design, off-target effects, insufficiently annotated datasets, and the intricate biological dynamics of plant-virus interactions. This paper summarizes the latest advancements in the incorporation of artificial intelligence (AI) methodologies, including machine learning and deep learning algorithms, into the CRISPR design and optimization framework. It examines how convolutional and recurrent neural networks, transformer architectures, and generative models like AlphaFold2, RoseTTAFold, and ESMFold can be used to predict protein structures, score sgRNAs, and model host-virus interactions. AI-enhanced methods have been proven to improve target specificity, Cas protein performance, and in silico validation. This paper aims to establish a foundation for next-generation genome editing strategies against plant viruses and promote the adoption of AI-powered CRISPR technologies in sustainable agriculture.
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Virus-Induced Genome Editing (VIGE): One Step Away from an Agricultural Revolution.
There is currently a worldwide trend towards deregulating the use of genome-edited plants. Virus-induced genome editing (VIGE) is a novel technique that utilizes viral vectors to transiently deliver clustered regularly interspaced short palindromic repeat (CRISPR) components into plant cells. It potentially allows us to obtain transgene-free events in any plant species in a single generation without in vitro tissue culture. This technology has great potential for agriculture and is already being applied to more than 14 plant species using more than 20 viruses. The main limitations of VIGE include insufficient vector capacity, unstable expression of CRISPR-associated (Cas) protein, plant immune reaction, host specificity, and reduced viral activity in meristem. Various solutions to these problems have been proposed, such as fusion of mobile elements, RNAi suppressors, novel miniature Cas proteins, and seed-borne viruses, but the final goal has not yet been achieved. In this review, the mechanism underlying the ability of different classes of plant viruses to transiently edit genomes is explained. It not only focuses on the latest achievements in virus-induced editing of crops but also provides suggestions for improving the technology. This review may serve as a source of new ideas for those planning to develop new approaches in VIGE.
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Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells.
Over 70 mutations in the transforming growth factor beta-induced (TGFBI) gene are associated with corneal dystrophies that impair vision. The R555W hotspot mutation is a major cause of granular corneal dystrophy type 1 (GCD1). Here, we evaluated the technical feasibility of CRISPR/Cas9-mediated editing of the R555W mutation in peripheral blood mononuclear cells (PBMCs) obtained from a patient with GCD1. Three single guide RNAs (sgRNA1-3) and matched single-stranded oligodeoxynucleotide donors (ssODN1-3) were designed and co-transfected into PBMCs. Transfected cells were enriched by flow cytometric sorting, with GFP-positive cells representing approximately 2-4% of the total electroporated population. Editing outcomes were initially screened using high-resolution melting (HRM) analysis, and the sgRNA3-ssODN3 combination identified as the most promising candidate was subsequently validated by next-generation sequencing (NGS). Sequencing revealed a homology-directed repair efficiency of 98.2% among GFP-positive sorted cells, demonstrating efficient and precise genome editing within the enriched population. Because PBMCs are not disease-relevant corneal epithelial cells and only genomic endpoints were assessed, the clinical applicability of this study is limited and the work should be considered a technical proof-of-concept. This framework supports optimization of CRISPR-based strategies prior to studies in biologically relevant corneal models.
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