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Found: 26 099
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Virus-induced plant genome editing.
Plant viruses have been engineered to express heterologous proteins and RNAs in plants for several decades. This viral system can now be applied to editing plant genomes. Virus vectors can deliver Cas proteins and guide RNAs, two key components of the CRISPR gene-editing system, into a plant cell without a complicated experimental procedure. In some cases, plant viruses move to meristematic cells and express gene-editing components in the cell, which results in the production of mutant seeds. Here, we focus on three main issues of the virus-induced genome editing (VIGE) technology in plants: (1) how to express the relatively large size of Cas proteins, (2) how to express guide RNA, and (3) how to increase the efficiency with which viruses are delivered into meristematic cells. We highlight recent advances in how plant virus vectors can be used efficiently in plant-genome editing.
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Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.
Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.
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CRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids.
CRISPR-Cas technology has revolutionized biological research and holds great therapeutic potential. Here, we review CRISPR-Cas systems and their latest developments with an emphasis on application to human cells. We also discuss how different CRISPR-based strategies can be used to accomplish a particular genome engineering goal. We then review how different CRISPR tools have been used in genome engineering of human stem cells in vitro, covering both the pluripotent (iPSC/ESC) and somatic adult stem cell fields and, in particular, 3D organoid cultures. Finally, we discuss the progress and challenges associated with CRISPR-based genome editing of human stem cells for therapeutic use.
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CRISPR/Cas9 gene-editing strategies in cardiovascular cells.
Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR-Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.
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In vivo CRISPR biosensing.
In vivo biosensing is essential for real-time monitoring of biological processes and disease progression within living organisms. Leveraging the programmable specificity and multifunctionality of CRISPR effectors, in vivo CRISPR-based biosensing has emerged as a powerful tool for highly sensitive and target-specific detection in complex physiological environments. This review presents the fundamental principles, design strategies, and bioanalytical applications of these advanced sensors, focusing on three key approaches: CRISPR-mediated highly efficient in vivo sequence recognition, CRISPR-driven trans-cleavage activity for signal amplification, and the use of base editors and prime editors for sensing-coupled genetic modulation. Critical design parameters-including delivery strategies, intracellular dynamics, and signal amplification mechanisms-are discussed in detail. We further highlight a broad range of applications, including in vivo DNA/RNA imaging, quantification of proteins and small molecules, gene-controlled drug release, dynamic signal recording, environmental response sensing, and lineage tracing in embryogenesis and tumor progression. The current challenges and outlining future directions are also discussed, underscoring the transformative potential of in vivo CRISPR biosensing in both fundamental biology and clinical translation.
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The potential application and challenge of powerful CRISPR/Cas9 system in cardiovascular research.
CRISPR/Cas9 is a precision-guided munition found in bacteria to fight against invading viruses. This technology has enormous potential applications, including altering genes in both somatic and germ cells, as well as generating knockout animals. Compared to other gene editing techniques such as zinc finger nucleases and TALENS, CRISPR/Cas9 is much easier to use and highly efficient. Importantly, the multiplex capacity of this technology allows multiple genes to be edited simultaneously. CRISPR/Cas9 also has the potential to prevent and cure human diseases. In this review, we wish to highlight some key points regarding the future prospect of using CRISPR/Cas9 as a powerful tool for cardiovascular research, and as a novel therapeutic strategy to treat cardiovascular diseases.
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Gene-Editing Technology Accelerates Cardiovascular Research: Clinical Applications Being Explored.
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Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.
The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.
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Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA.
The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.
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CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters.
The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We first performed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences for enhancing the heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.
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