Detection of CRISPR-Cas9-Mediated Mutations Using a Carbon Nanotube-Modified Electrochemical Genosensor

The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5′-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes.

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First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill

Frontiers in Plant Science ◽

10.3389/fpls.2021.728516 ◽

2021 ◽

Vol 12 ◽

Author(s):

Vera Pavese ◽

Andrea Moglia ◽

Elena Corredoira ◽

Mª Teresa Martínez ◽

Daniela Torello Marinoni ◽

...

Keyword(s):

Somatic Embryos ◽

Gene Editing ◽

Genome Engineering ◽

High Efficiency ◽

Chlorophyll Biosynthesis ◽

Point Mutations ◽

Castanea Sativa ◽

Visual Assessment ◽

European Chestnut ◽

Selection Medium

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

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Precise genome engineering inDrosophilausing prime editing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021996118 ◽

2020 ◽

Vol 118 (1) ◽

pp. e2021996118

Author(s):

Justin A. Bosch ◽

Gabriel Birchak ◽

Norbert Perrimon

Keyword(s):

Gene Function ◽

Mammalian Cells ◽

Genome Engineering ◽

Cultured Cells ◽

Germ Line ◽

Point Mutations ◽

Model Organism ◽

Model Organisms ◽

Marker Genes ◽

Germ Line Transmission

Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organismDrosophila melanogasterand developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes,ebony,white, andforked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit inebonyto 36% of progeny. Our results suggest that prime editing is a useful system inDrosophilato study gene function, such as engineering precise point mutations, deletions, or epitope tags.

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Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02565-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Manoj Kumar K. Azhagiri ◽

Prathibha Babu ◽

Vigneshwaran Venkatesan ◽

Saravanabhavan Thangavel

Keyword(s):

Gene Therapy ◽

Gene Editing ◽

Genome Engineering ◽

Point Mutations ◽

Target Cells ◽

Gene Manipulation ◽

Hematopoietic Stem ◽

Knock Out ◽

Base Editing ◽

Stem And Progenitor Cells

AbstractThe advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.

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Precise genome engineering in Drosophila using prime editing

10.1101/2020.08.05.232348 ◽

2020 ◽

Cited By ~ 3

Author(s):

Justin A. Bosch ◽

Gabriel Birchak ◽

Norbert Perrimon

Keyword(s):

Gene Function ◽

Mammalian Cells ◽

Genome Engineering ◽

Cultured Cells ◽

Germ Line ◽

Point Mutations ◽

Model Organism ◽

Model Organisms ◽

Marker Genes ◽

Germ Line Transmission

AbstractPrecise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely installed premature stop codons in three classical visible marker genes, ebony, white, and forked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ line transmission of a precise edit in ebony to ~50% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.

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Strategies for Bacteriophage T5 Mutagenesis: Expanding the Toolbox for Phage Genome Engineering

Frontiers in Microbiology ◽

10.3389/fmicb.2021.667332 ◽

2021 ◽

Vol 12 ◽

Author(s):

Luis Ramirez-Chamorro ◽

Pascale Boulanger ◽

Ombeline Rossier

Keyword(s):

Gene Editing ◽

Genome Engineering ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Phage Genome ◽

Essential Gene ◽

Point Mutations ◽

Antibacterial Properties ◽

Protein Coding ◽

Phage Gene

Phage genome editing is crucial to uncover the molecular mechanisms of virus infection and to engineer bacteriophages with enhanced antibacterial properties. Phage genetic engineering relies mostly on homologous recombination (HR) assisted by the targeted elimination of wild-type phages by CRISPR-Cas nucleases. These strategies are often less effective in virulent bacteriophages with large genomes. T5 is a virulent phage that infects Escherichia coli. We found that CRISPR-Cas9 system (type II-A) had ununiform efficacies against T5, which impairs a reliable use of CRISPR-Cas-assisted counterselection in the gene editing of T5. Here, we present alternative strategies for the construction of mutants in T5. Bacterial retroelements (retrons) proved to be efficient for T5 gene editing by introducing point mutations in the essential gene A1. We set up a protocol based on dilution-amplification-screening (DAS) of phage pools for mutant enrichment that was used to introduce a conditional mutation in another essential gene (A2), insert a new gene (lacZα), and construct a translational fusion of a late phage gene with a fluorescent protein coding gene (pb10-mCherry). The method should be applicable to other virulent phages that are naturally resistant to CRISPR/Cas nucleases.

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Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽

10.3390/biology10060530 ◽

2021 ◽

Vol 10 (6) ◽

pp. 530

Author(s):

Marlo K. Thompson ◽

Robert W. Sobol ◽

Aishwarya Prakash

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

Genome Stability ◽

Gene Editing ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Specific Gene ◽

Target Sequence ◽

Transcription Activator ◽

Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

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Electrochemical Investigation of Curcumin–DNA Interaction by Using Hydroxyapatite Nanoparticles–Ionic Liquids Based Composite Electrodes

Materials ◽

10.3390/ma14154344 ◽

2021 ◽

Vol 14 (15) ◽

pp. 4344

Author(s):

Merve Uca ◽

Ece Eksin ◽

Yasemin Erac ◽

Arzum Erdem

Keyword(s):

Electrochemical Impedance ◽

Differential Pulse ◽

Dna Interaction ◽

Composite Electrodes ◽

Hydroxyapatite Nanoparticles ◽

Graphite Electrodes ◽

X Ray ◽

Pulse Voltammetry ◽

Polymerase Chain

Hydroxyapatite nanoparticles (HaP) and ionic liquid (IL) modified pencil graphite electrodes (PGEs) are newly developed in this assay. Electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and cyclic voltammetry (CV) were applied to examine the microscopic and electrochemical characterization of HaP and IL-modified biosensors. The interaction of curcumin with nucleic acids and polymerase chain reaction (PCR) samples was investigated by measuring the changes at the oxidation signals of both curcumin and guanine by differential pulse voltammetry (DPV) technique. The optimization of curcumin concentration, DNA concentration, and the interaction time was performed. The interaction of curcumin with PCR samples was also investigated by gel electrophoresis.

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A Label-Free Electrochemical Aptasensor for the Rapid Detection of Tetracycline Based on Ordered Mesoporous Carbon–Fe3O4

Australian Journal of Chemistry ◽

10.1071/ch17503 ◽

2018 ◽

Vol 71 (3) ◽

pp. 170 ◽

Cited By ~ 2

Author(s):

Xuejia Zhan ◽

Guangzhi Hu ◽

Thomas Wagberg ◽

Dongwei Zhang ◽

Pei Zhou

Keyword(s):

Photoelectron Spectroscopy ◽

Mesoporous Carbon ◽

Physical Adsorption ◽

Differential Pulse ◽

Ordered Mesoporous Carbon ◽

Label Free ◽

X Ray Diffraction ◽

X Ray ◽

Ordered Mesoporous ◽

Modification Procedure

A novel aptasensor based on a tetracycline (TET) aptamer immobilized by physical adsorption on an ordered mesoporous carbon–Fe3O4 (OMC-Fe3O4)-modified screen-printed electrode surface was successfully fabricated. OMC-Fe3O4 was characterized by scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The modification procedure of the aptasensor was characterized by cyclic voltammetry. Interaction between the TET aptamer and target was determined by differential pulse voltammetry. Under optimal conditions, the proposed aptasensor exhibited good electrochemical sensitivity to TET in a concentration range of 5 nM to 10 μM, with a detection limit of 0.8 nM (S/N = 3). This aptasensor exhibited satisfactory specificity, reproducibility, and stability.

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