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Emerging techniques of CRISPR/Cas system in antiviral therapy and diagnostics: Applications, limitations, and translational perspectives.
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.
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CRISPR/Cas9 Technology: Applications and Human Disease Modeling.
The CRISPR/Cas9 system development has revolutionized the field of genome engineering through the efficient creation of targeted breaks in the DNA of almost any organism and cell type, opening an avenue for a wide range of applications in biomedical research and medicine. Apart from gene edition through knock-in or knock-out approaches, CRISPR/Cas9 technology has been used for many other purposes, including regulation of endogenous gene expression, epigenome editing, live-cell imaging of chromosomal loci, edition of RNA and high-throughput screening. With all those technological improvements, CRISPR/Cas9 system has broadened the number of alternatives for studying gene function and the generation of more accurate disease models. Although many mechanistic questions remain to be answered and several challenges have yet to be addressed, the use of CRISPR/Cas9-based genome engineering technologies will increase our knowledge of disease processes and their treatment in the near future.
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Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9.
Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.
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[Genetic manipulation in zebrafish].
The increasing number of genetic manipulation approaches and high-resolution live imaging technique applied in zebrafish have propelled the rise of this organism as a mainstream model for developmental biology and human diseases studies. Zebrafish has many advantages for functional genomics analysis, allowing for easy, cheap and fast functional characterization of novel genes in the vertebrate genome. Here we provide an overview of the principles of genetic manipulation in zebrafish, such as Ethylnitrosourea (ENU) mutagenesis, insertional mutagenesis, gene trapping mutagenesis, Morpholino mediated gene knockdown, targeting induced local lesions in genomes (TILLING), genome editing with engineered nucleases ZFN (Zinc finger nuclease), TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 system, and transgenic methods used in zebrafish.
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SuperDecode: An integrated toolkit for analyzing mutations induced by genome editing.
Genome editing using CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) or other systems has become a cornerstone of numerous biological and applied research fields. However, detecting the resulting mutations by analyzing sequencing data remains time consuming and inefficient. In response to this issue, we designed SuperDecode, an integrated software toolkit for analyzing editing outcomes using a range of sequencing strategies. SuperDecode comprises three modules, DSDecodeMS, HiDecode, and LaDecode, each designed to automatically decode mutations from Sanger, high-throughput short-read, and long-read sequencing data, respectively, from targeted PCR amplicons. By leveraging specific strategies for constructing sequencing libraries of pooled multiple amplicons, HiDecode and LaDecode facilitate large-scale identification of mutations induced by single or multiplex target-site editing in a cost-effective manner. We demonstrate the efficacy of SuperDecode by analyzing mutations produced using different genome editing tools (CRISPR/Cas, base editing, and prime editing) in different materials (diploid and tetraploid rice and protoplasts), underscoring its versatility in decoding genome editing outcomes across different applications. Furthermore, this toolkit can be used to analyze other genetic variations, as exemplified by its ability to estimate the C-to-U editing rate of the cellular RNA of a mitochondrial gene. SuperDecode offers both a standalone software package and a web-based version, ensuring its easy access and broad compatibility across diverse computer systems. Thus, SuperDecode provides a comprehensive platform for analyzing a wide array of mutations, advancing the utility of genome editing for scientific research and genetic engineering.
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CRISPR-Cas9: a new and promising player in gene therapy.
First introduced into mammalian organisms in 2013, the RNA-guided genome editing tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) offers several advantages over conventional ones, such as simple-to-design, easy-to-use and multiplexing (capable of editing multiple genes simultaneously). Consequently, it has become a cost-effective and convenient tool for various genome editing purposes including gene therapy studies. In cell lines or animal models, CRISPR-Cas9 can be applied for therapeutic purposes in several ways. It can correct the causal mutations in monogenic disorders and thus rescue the disease phenotypes, which currently represents the most translatable field in CRISPR-Cas9-mediated gene therapy. CRISPR-Cas9 can also engineer pathogen genome such as HIV for therapeutic purposes, or induce protective or therapeutic mutations in host tissues. Moreover, CRISPR-Cas9 has shown potentials in cancer gene therapy such as deactivating oncogenic virus and inducing oncosuppressor expressions. Herein, we review the research on CRISPR-mediated gene therapy, discuss its advantages, limitations and possible solutions, and propose directions for future research, with an emphasis on the opportunities and challenges of CRISPR-Cas9 in cancer gene therapy.
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Challenges and Opportunities with CRISPR-Based Genome Editing in Legume Crops.
Over the last couple of decades, tremendous progress has been made in legume genomics. Genomics information generated for legume crops is being explored through molecular breeding and transgenic approaches. However, the gap between knowledge generation and its utilization is increasing. In this regard, recent developments in genome editing techniques provide an excellent opportunity to utilize the available knowledge for the improvement of legume crops. This review highlights recent developments with Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome-editing approaches, including Cas variants/orthologs and Protospacer adjacent motif-less (PAMless) Genome Editing, multiplex genome editing, base editing, prime editing, transcriptional regulation, methylome editing, and DNA-free editing methods. Furthermore, the applications of non-homologous end joining (NHEJ) and homology-directed repair (HDR)- based editing, are addressed which enable targeted and precise genomic modifications. Moreover, virus-mediated genome editing, in planta transformation, and mobile guide RNAs are increasingly being leveraged to enhance the efficiency and heritability of genome editing. Additionally, the role of artificial intelligence in guide RNA design, off-target prediction, and the development of novel Cas variants is also discussed, which can speed up the legume improvement. This article highlights the successful examples of efforts utilizing CRISPR/Cas9 for the development of legume crops with biotic and abiotic stress tolerance, desirable plant architecture, improved nutrient uptake, and enhanced yield and quality. The biggest limitation in the genome editing of legume crops is their recalcitrance to both transformation and tissue culture. This article discusses how this particular limitation can be addressed in the context of genome editing of legume crops. Finally, the possibilities of integrating these recently developed tools with translational breeding have also been discussed, which will facilitate the legume production for sustainable agriculture and food security.
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Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
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Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori.
Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or single-base deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.
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CRISPR Therapeutics for Duchenne Muscular Dystrophy.
Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder with a prevalence of approximately 1 in 3500-5000 males. DMD manifests as childhood-onset muscle degeneration, followed by loss of ambulation, cardiomyopathy, and death in early adulthood due to a lack of functional dystrophin protein. Out-of-frame mutations in the dystrophin gene are the most common underlying cause of DMD. Gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising therapeutic for DMD, as it can permanently correct DMD mutations and thus restore the reading frame, allowing for the production of functional dystrophin. The specific mechanism of gene editing can vary based on a variety of factors such as the number of cuts generated by CRISPR, the presence of an exogenous DNA template, or the current cell cycle stage. CRISPR-mediated gene editing for DMD has been tested both in vitro and in vivo, with many of these studies discussed herein. Additionally, novel modifications to the CRISPR system such as base or prime editors allow for more precise gene editing. Despite recent advances, limitations remain including delivery efficiency, off-target mutagenesis, and long-term maintenance of dystrophin. Further studies focusing on safety and accuracy of the CRISPR system are necessary prior to clinical translation.
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