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

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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CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research

10.1101/017384 ◽

2015 ◽

Author(s):

Fillip Port ◽

Nadine Muschalik ◽

Simon L Bullock

Keyword(s):

Gene Editing ◽

Genome Engineering ◽

High Efficiency ◽

Basic Research ◽

Evidence Based ◽

Gene Drive ◽

Highly Active ◽

Genome Modification ◽

Target Sites ◽

Gene Drives

CRISPR/Cas technology allows rapid, site-specific genome modification in a wide variety of organisms. CRISPR components produced by integrated transgenes have been shown to mutagenise some genomic target sites in Drosophila melanogaster with high efficiency, but whether this is a general feature of this system remains unknown. Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk.

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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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Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene

New Forests ◽

10.1007/s11056-016-9537-5 ◽

2016 ◽

Vol 47 (5) ◽

pp. 669-684 ◽

Cited By ~ 15

Author(s):

E. Corredoira ◽

M. C. San José ◽

A. M. Vieitez ◽

I. Allona ◽

C. Aragoncillo ◽

...

Keyword(s):

Somatic Embryos ◽

Castanea Sativa ◽

Agrobacterium Mediated Transformation ◽

European Chestnut ◽

Castanea Sativa Mill ◽

Endochitinase Gene

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A minimal CRISPR-Cas3 system for genome engineering

10.1101/860999 ◽

2019 ◽

Author(s):

Bálint Csörgő ◽

Lina M. León ◽

Ilea J. Chau-Ly ◽

Alejandro Vasquez-Rifo ◽

Joel D. Berry ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Genome Engineering ◽

High Efficiency ◽

Genetic Manipulation ◽

Point Mutations ◽

Type I ◽

Homology Directed Repair ◽

Large Deletions ◽

System Type ◽

Genome Scale

AbstractCRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an “all-in-one” vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

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Various Aspects of a Gene Editing System—CRISPR–Cas9

International Journal of Molecular Sciences ◽

10.3390/ijms21249604 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9604

Author(s):

Edyta Janik ◽

Marcin Niemcewicz ◽

Michal Ceremuga ◽

Lukasz Krzowski ◽

Joanna Saluk-Bijak ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Genome Engineering ◽

High Efficiency ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Guide Rna ◽

Adaptive Immune ◽

Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

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

Biosensors ◽

10.3390/bios11010017 ◽

2021 ◽

Vol 11 (1) ◽

pp. 17

Author(s):

Ezgi Kivrak ◽

Tekle Pauzaite ◽

Nikki A. Copeland ◽

John G. Hardy ◽

Pinar Kara ◽

...

Keyword(s):

Carbon Nanotube ◽

Gene Editing ◽

Genome Engineering ◽

Point Mutations ◽

Differential Pulse ◽

Model Organisms ◽

Target Sequence ◽

Label Free ◽

Graphite Electrodes ◽

Guanine Oxidation

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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Genetic transformation of European chestnut somatic embryos with a native thaumatin-like protein (CsTL1) gene isolated from Castanea sativa seeds

Tree Physiology ◽

10.1093/treephys/tps098 ◽

2012 ◽

Vol 32 (11) ◽

pp. 1389-1402 ◽

Cited By ~ 19

Author(s):

E. Corredoira ◽

S. Valladares ◽

I. Allona ◽

C. Aragoncillo ◽

A. M. Vieitez ◽

...

Keyword(s):

Genetic Transformation ◽

Somatic Embryos ◽

Castanea Sativa ◽

European Chestnut

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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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CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽

10.1093/genetics/80.4.667 ◽

1975 ◽

Vol 80 (4) ◽

pp. 667-678

Author(s):

Mary Lee S Ledbetter ◽

Rollin D Hotchkiss

Keyword(s):

High Efficiency ◽

Point Mutations ◽

Resistant Mutant ◽

Chromosomal Marker ◽

Structural Abnormality ◽

C Cells ◽

Wild Type ◽

Chromosomal Locus ◽

Low Efficiency ◽

Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

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