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Virus-induced genome editing: toward crop breeding applications.
CRISPR-Cas-based genome editing has revolutionized precise genome manipulation in plants, yet its practical application is still constrained by the inefficient delivery of editing reagents across different genotypes. Plant viruses are promising vehicles for delivering genome-editing components, bypassing plant transformation and/or tissue culture. Virus-induced genome editing (VIGE) has provided powerful tools for achieving heritable edits in model plants such as Arabidopsis thaliana and Nicotiana benthamiana. VIGE has now progressed from proof-of-concept to practical applications in agricultural crops. Notably, a recent breakthrough in VIGE in tiller has successfully achieved heritable genome editing in hexaploid wheat. This review outlines the latest advances in VIGE across diverse plant species, highlights its potential for crop improvement, and discusses future research directions.
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De novo screening of disease-resistant genes from the chromosome-level genome of rare minnow using CRISPR-cas9 random mutation.
BACKGROUND: Mutants are important for the discovery of functional genes and creation of germplasm resources. Mutant acquisition depends on the efficiency of mutation technology and screening methods. CRISPR-Cas9 technology is an efficient gene editing technology mainly used for editing a few genes or target sites, which has not been applied for the construction of random mutant libraries and for the de novo discovery of functional genes. RESULTS: In this study, we first sequenced and assembled the chromosome-level genome of wild-type rare minnow (Gobiocypris rarus) as a susceptible model of hemorrhagic disease, obtained a 956.05 Mb genome sequence, assembled the sequence into 25 chromosomes, and annotated 26,861 protein-coding genes. Thereafter, CRISPR-Cas9 technology was applied to randomly mutate the whole genome of rare minnow with the conserved bases (TATAWAW and ATG) of the promoter and coding regions as the target sites. The survival rate of hemorrhagic disease in the rare minnow gradually increased from 0% (the entire wild-type population died after infection) to 38.24% (F3 generation). Finally, 7 susceptible genes were identified via genome comparative analysis and cell-level verification based on the rare minnow genome. CONCLUSIONS: The results provided the genomic resources for wild-type rare minnow, and confirmed that the random mutation system designed using CRISPR-Cas9 technology in this study is simple and efficient and is suitable for the de novo discovery of functional genes and creation of a germplasm resource related to qualitative traits.
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Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a.
The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.
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Overview of gene editing strategies against HBV.
254 million people currently live with chronic hepatitis B virus (HBV) infection, with over 1 million deaths annually due to complications such as cirrhosis and hepatocellular carcinoma. Although current direct-acting antivirals suppress HBV replication, they do not eliminate the virus and rarely lead to HBV functional cure, defined as the loss of serum hepatitis B surface antigen (HBsAg) and DNA. A major barrier to achieving HBV functional cure is the HBV covalently closed circular DNA minichromosome (cccDNA), which hides from the immune system in the nucleus of an infected cell, and is very stable. Another barrier is integration of incomplete HBV genomes into the host genome, which is the main source of HBsAg in later disease stages, and is difficult to target without impacting the human genome. New direct-acting antivirals are required that target different stages of the HBV replication cycle, including the HBV cccDNA and integrated DNA to improve rates of functional cure. The development of gene editing tools provides an opportunity to develop novel therapies that target the HBV cccDNA, integrated DNA and HBV RNA. This review explores the different gene editing tools that have been used to target the HBV cccDNA, integrated DNA and RNA.
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CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art.
In recent years, a variety of genetic tools have been developed and applied to various filamentous fungi, which are widely applied in agriculture and the food industry. However, the low efficiency of gene targeting has for many years hampered studies on functional genomics in this important group of microorganisms. The emergence of CRISPR/Cas9 genome-editing technology has sparked a revolution in genetic research due to its high efficiency, versatility, and easy operation and opened the door for the discovery and exploitation of many new natural products. Although the application of the CRISPR/Cas9 system in filamentous fungi is still in its infancy compared to its common use in E. coli, yeasts, and mammals, the deep development of this system will certainly drive the exploitation of fungal diversity. In this review, we summarize the research progress on CRISPR/Cas9 systems in filamentous fungi and finally highlight further prospects in this area.
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Novel Approaches of CRISPR-Cas Technology in Airway Diseases.
Clustered regularly interspaced palindromic repeats (CRISPR) technique plays a vital role in preclinical modelling of many respiratory diseases. Diseases such as chronic obstructive pulmonary disease (COPD), asthma, acute tracheal bronchitis, pneumonia, tuberculosis, lung cancer, and influenza infection continue to significantly impact human health. CRISPR associated (Cas) proteins, isolated from the immune system of prokaryotes, are one component of a very useful technique to manipulate gene sequences or editing and gene expression with significant implications for respiratory research in the field of molecular biology. CRISPR technology is a promising tool that is easily adaptable for specific editing of DNA sequences of interest with a goal towards modifying or eliminating gene function. Among its many potential applications, CRISPR can be applied to correcting genetic defects as well as for therapeutic approaches for treatment. This review elucidates recent advances in CRISPR-Cas technology in airway diseases.
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Opportunities and challenges with CRISPR-Cas mediated homologous recombination based precise editing in plants and animals.
We summarise recent advancements to achieve higher homologous recombination based gene targeting efficiency in different animals and plants. The genome editing has revolutionized the agriculture and human therapeutic sectors by its ability to create precise, stable and predictable mutations in the genome. It depends upon targeted double-strand breaks induction by the engineered endonucleases, which then gets repaired by highly conserved endogenous DNA repair mechanisms. The repairing could be done either through non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The HDR-based editing can be applied for precise gene targeting such as insertion of a new gene, gene replacement and altering of the regulatory sequence of a gene to control the existing protein expression. However, HDR-mediated editing is considered challenging because of lower efficiency in higher eukaryotes, thus, preventing its widespread application. This article reviews the recent progress of HDR-mediated editing and discusses novel strategies such as cell cycle synchronization, modulation of DNA damage repair factors, engineering of Cas protein favoring HDR and CRISPR-Cas reagents delivery methods to improve efficiency for generating knock-in events in both plants and animals. Further, multiplexing of described methods may be promising towards achieving higher donor template-assisted homologous recombination efficiency at the target locus.
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Advancing sensing technology with CRISPR: From the detection of nucleic acids to a broad range of analytes - A review.
The CRISPR/Cas technology, derived from an adaptive immune system in bacteria, has been awarded the Nobel Prize in Chemistry in 2020 for its success in gene editing. Increasing reports reveal that CRISPR/Cas technology has a wide scope of applications and it could be incorporated into biosensors for detecting critical analytes. CRISPR-powered biosensors have attracted significant research interest due to their advantages including high accuracy, good specificity, rapid response, and superior integrity. Now the CRISPR technology is not only admirable in nucleic acid monitoring, but also promising for other kinds of biomarkers' detection, including metal ions, small molecules, peptides, and proteins. Therefore, it is of great worth to explore the prospect, and summarize the strategies in applying CRISPR technology for the recognition of a broad range of targets. In this review, we summarized the strategies of CRISPR biosensing for non-nucleic-acid analytes, the latest development of nucleic acid detection, and proposed the challenges and outlook of CRISPR-powered biosensors.
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Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro.
Corneal wound healing is influenced by many factors including transcriptional co-repressors and co-activators. Interactions of co-activators and co-repressors with Smads influence mechanistic loop facilitating transcription of alpha-smooth muscle actin (α-SMA), a key profibrotic gene, in corneal repair. The role of a transcriptional repressor, 5'TG3'-interacting factor (TGIF), in the regulation of α-SMA and myofibroblast formation in the cornea was shown previously by our group. This study tested a hypothesis if TGIF1 gene editing via CRISPR/Cas9 can ease myofibroblast formation in the cornea using an in vitro model. Primary human corneal stromal fibroblasts (hCSFs) generated from donor corneas received gene-editing plasmid facilitating loss (CRISPR/Cas9 knockout) or gain (CRISPR activation) of TGIF function by UltraCruz transfection reagent. Phase-contrast microscopy, immunoblotting, immunocytochemistry and quantitative polymerase chain reaction (qPCR) were used to measure levels of myofibroblast profibrotic genes (α-SMA, fibronectin, Collagen-I, and Collagen-IV) in hCSFs lacking or overexpressing TGIF1 after growing them in± transforming growth factor beta1 (TGF-β1) under serum-free conditions. The CRISPR-assisted TGIF1 activation (gain of function) in hCSFs demonstrated significantly decreased myofibroblast formation and messenger ribonucleic acid (mRNA) and protein levels of profibrotic genes. Conversely, CRISPR/Cas9-assisted TGIF knockdown (loss of function) in hCSFs demonstrated no significant change in the levels of myofibroblast formation or profibrotic genes under similar conditions. These results suggest that TGIF gene-editing approach can be employed to modulate the transcriptional activity of α-SMA in controlling pathological and promoting physiological wound healing in an injured cornea.
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Efficient Intracellular Delivery of CRISPR-Cas Ribonucleoproteins through Receptor Mediated Endocytosis.
We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.
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