Accurate Direct PCR with Arabidopsis and rice

Methods for PCR that avoid costly and time consuming plant DNA purification have not been widely adopted, partly because their efficacy is unclear. Here, we compare different sampling methods for Direct PCR in Arabidopsis and rice. CutTip, stabbing a pipette tip into a plant organ and depositing the tip of the pipette tip into the reaction buffer, yielded high accuracy for genotyping and detection of CRISPR-induced mutations. This did not require visible tissue fragments in the reactions. We demonstrate the usefulness of this method in sampling many locations within a single plant to identify a rare CRISPR-mutated sector. These methods are simple, inexpensive and can help address the challenge of genotyping and genome editing at different scales with high accuracy. The methods also simplify the application of PCR in the field.

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Workflow for generating HMW plant DNA for third generation sequencing with high N50 and high accuracy v1 (protocols.io.bafmibk6)

protocols.io ◽

10.17504/protocols.io.bafmibk6 ◽

2019 ◽

Author(s):

Patrick Driguez ◽

Karen Carty ◽

Alexander Putra ◽

Luca Ermini

Keyword(s):

High Accuracy ◽

Third Generation ◽

Plant Dna ◽

Third Generation Sequencing ◽

Generation Sequencing

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Concepts of Molecular Plant Breeding and Genome Editing

Advances in Environmental Engineering and Green Technologies - Molecular Plant Breeding and Genome Editing Tools for Crop Improvement ◽

10.4018/978-1-7998-4312-2.ch001 ◽

2021 ◽

pp. 1-15

Keyword(s):

Plant Breeding ◽

Genome Editing ◽

Genetic Linkage ◽

Selection Process ◽

Crop Improvement ◽

Zinc Finger Nucleases ◽

Induced Mutations ◽

Transcription Activator ◽

Targeted Gene Editing ◽

Breeding Techniques

Traditional plant breeding depends on spontaneous and induced mutations available in the crop plants. Such mutations are rare and occur randomly. By contrast, molecular breeding and genome editing are advanced breeding techniques that can enhance the selection process and produce precisely targeted modifications in any crop. Identification of molecular markers, based on SSRs and SNPs, and the availability of high-throughput (HTP) genotyping platforms have accelerated the process of generating dense genetic linkage maps and thereby enhanced application of marker-assisted breeding for crop improvement. Advanced molecular biology techniques that facilitate precise, efficient, and targeted modifications at genomic loci are termed as “genome editing.” The genome editing tools include “zinc-finger nucleases (ZNFs),” “transcription activator-like effector nucleases (TALENs),” oligonucleotide-directed mutagenesis (ODM), and “clustered regularly interspersed short palindromic repeats (CRISPER/Cas) system,” which can be used for targeted gene editing. Concepts of molecular plant breeding and genome editing systems are presented in this chapter.

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Co-existence of Positive and Negative Heterosis in a Single Plant Organ

Nature ◽

10.1038/1691054a0 ◽

1952 ◽

Vol 169 (4312) ◽

pp. 1054-1055 ◽

Cited By ~ 2

Author(s):

RONALD MELVILLE

Keyword(s):

Single Plant ◽

Plant Organ

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Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome

10.1101/267690 ◽

2018 ◽

Cited By ~ 2

Author(s):

Eirini M Kallimasioti-Pazi ◽

Keerthi Thelakkad Chathoth ◽

Gillian C Taylor ◽

Alison Meynert ◽

Tracy Ballinger ◽

...

Keyword(s):

Genome Editing ◽

Imprinted Genes ◽

Induced Mutations ◽

Permeable Barrier ◽

Homology Directed Repair ◽

Confounding Variables ◽

Experimental Parameters ◽

Allele Specific ◽

And Function ◽

Mutagenic Dna Repair

AbstractCRISPR-Cas9 genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem. Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of Cas9-induced mutations can be attributed unequivocally to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. Surprisingly, the outcome of mutagenic DNA repair was independent of chromatin state, with similar efficiencies of homology directed repair and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint of CRISPR-Cas9 genome editing, and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

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31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab31 ◽

2015 ◽

Vol 27 (1) ◽

pp. 108

Author(s):

H. Matsunari ◽

M. Watanabe ◽

K. Nakano ◽

A. Uchikura ◽

Y. Asano ◽

...

Keyword(s):

Somatic Cell ◽

Genome Editing ◽

Production Efficiency ◽

Gene Knockout ◽

Genetically Modified ◽

Zinc Finger Nucleases ◽

International Institute ◽

Induced Mutations ◽

Transcription Activator

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

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CRISPR/Cas9-mediated genome editing in a reef-building coral

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722151115 ◽

2018 ◽

Vol 115 (20) ◽

pp. 5235-5240 ◽

Cited By ~ 36

Author(s):

Phillip A. Cleves ◽

Marie E. Strader ◽

Line K. Bay ◽

John R. Pringle ◽

Mikhail V. Matz

Keyword(s):

Genome Editing ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Mutant Gene ◽

Mutation Induction ◽

Induced Mutations ◽

Important Species ◽

Ribonucleoprotein Complexes ◽

Genes Encoding ◽

Green Fluorescent

Reef-building corals are critically important species that are threatened by anthropogenic stresses including climate change. In attempts to understand corals’ responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating a variety of hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals. Here, we demonstrate efficient genome editing using the CRISPR/Cas9 system in the coral Acropora millepora. We targeted the genes encoding fibroblast growth factor 1a (FGF1a), green fluorescent protein (GFP), and red fluorescent protein (RFP). After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, we detected induced mutations in the targeted genes using changes in restriction-fragment length, Sanger sequencing, and high-throughput Illumina sequencing. We observed mutations in ∼50% of individuals screened, and the proportions of wild-type and various mutant gene copies in these individuals indicated that mutation induction continued for at least several cell cycles after injection. Although multiple paralogous genes encoding green fluorescent proteins are present in A. millepora, appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because A. millepora larvae can be induced to settle and begin colony formation in the laboratory, CRISPR/Cas9-based gene editing should allow rigorous tests of gene function in both larval and adult corals.

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CRISPR Pioneers Win 2020 Nobel Prize for Chemistry

Iranian Journal of Public Health ◽

10.18502/ijph.v49i12.4800 ◽

2020 ◽

Author(s):

Dariush D. FARHUD ◽

Marjan ZARIF-YEGANEH

Keyword(s):

Clinical Practice ◽

Human Genome ◽

Genome Editing ◽

Nobel Prize ◽

Gene Editing ◽

High Accuracy ◽

Recent Advances ◽

Programmable Nucleases

Over the last few years, the development of genome editing has revolutionized research on the human genome. Recent advances in developing programmable nucleases, such as meganucleases, ZFNs, TALENs and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas, has greatly expedited the progress of gene editing from concept to clinical practice. The CRISPR has advantages over other nuclease-based genome editing tools due to its high accuracy, efficiency, and strong specificity. Eight years after CRISPR application for human genome edition by Emmanuelle Charpentier and Jennifer A. Doudna, the 2020 Nobel Prize in Chemistry has been jointly given to them for development of CRISPR-Cas9 gene editing, allows scientists to precisely cut and edit of DNA.

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Pengembangan Protokol Isolasi DNA Genom Tanaman Durian Dengan Menggunakan Modifikasi Bufer CTAB

TECHNO JURNAL PENELITIAN ◽

10.33387/tk.v6i02.567 ◽

2018 ◽

Vol 6 (02) ◽

pp. 33

Author(s):

Sundari Sundari

Keyword(s):

Efficient Method ◽

Genomic Dna ◽

Dna Isolation ◽

Dna Purification ◽

Fruit Crops ◽

Purification Step ◽

High Quality ◽

Plant Dna ◽

Genomic Dna Isolation

AbstrakProtokol dan metode sederhana, efisien untuk isolasi DNA genom tanaman durian yang banyak mengandung phenol dan residu polisakarida telah dihasilkan. Pada penelitian ini, digunakan protokol isolasi DNA tumbuhan dengan metode CTAB yang dimodifikasi sebagai protokol yang efisien untuk membuang polisakarida, phenol dan lendir yang sangat melimpah pada tanaman durian. Obyek penelitian ini terdiri dari protocol CTAB yang dimodifikasi tahap inkubasi dan presipitasi pemurnian DNA genom dari phenol dan polisakarida. Perbandingan 2 protokol isolasi DNA durian dengan CTAB standard an CTAB modifikasi menunjukkan bahwa metode CTAB modifikasi menghasilkan whole genom durian cukup murni rata rata 1,99 dan berhasil diamplifikasi dengan PCR-RAPD.Kata kunci: isolasi, DNA, polisakarida, CTAB, modifikasi, . AbstractThe simple and efficient method for genomic DNA isolation protochol from durian , its woody fruit crops containing high polysaccharide levels has been described here. In the present study, using modified CTAB for plant DNA isolation protocols were studied for removing the highly concentrated polysaccharides from genomic DNA of woody fruit crops.This method involves the modified CTAB at the incubate and precipitate procedure employing DNA purification step to remove polysaccharides and phenol residu. Compared with the two studied DNA isolation protocols of durian using standart CTAB and modified CTAB the everage yield high quality DNA whole genom is 1,99 purity and DNA was suitable for PCR and RAPD analyses.Keyword: isolation, DNA, polysaccharides, phenol residu, CTAB

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Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in Vitis vinifera tissues

OENO One ◽

10.20870/oeno-one.2014.48.4.1697 ◽

2014 ◽

Vol 48 (4) ◽

pp. 261 ◽

Cited By ~ 1

Author(s):

Katia Gindro ◽

Nicole Lecoultre ◽

Luca Molino ◽

Jean-Pierre De Joffrey ◽

Sylvain Schnee ◽

...

Keyword(s):

Vitis Vinifera ◽

Downy Mildew ◽

Fungal Pathogens ◽

Pcr Amplification ◽

Microscopic Analysis ◽

Grey Mould ◽

Dna Purification ◽

Direct Pcr ◽

Fungal Propagules ◽

Pcr Method

Aims: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples.Methods and results: Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specifie for Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Vitis vinifera. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for P. viticola to one conidium for E. necator. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of E. necator and B. cinerea in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of P. viticola.Conclusion: A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew.Significance and impact of the study: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations.

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