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Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9

Authors: Florian Hahn
Florian HahnAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4806
, Marion Eisenhut
Marion EisenhutAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4807
, Otho Mantegazza
Otho MantegazzaAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4808
 and Andreas P. M. Weber
Andreas P. M. WeberAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
For correspondence: Andreas.Weber@uni-duesseldorf.de
Bio-protocol author page: a4809
,  date: 7/5/2017, 133 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2384.

[Abstract] The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for ...

Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses

Authors: Toshihisa Nomura
Toshihisa NomuraAffiliation: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, Japan
For correspondence: toshihisa.nomura@riken.jp
Bio-protocol author page: a4737
 and Hitoshi Sakakibara
Hitoshi SakakibaraAffiliation 1: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, Japan
Affiliation 2: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Bio-protocol author page: a4739
,  date: 6/20/2017, 273 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2359.

[Abstract] RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura ...

Determining Genome Size from Spores of Seedless Vascular Plants

Authors: Li-Yaung Kuo
Li-Yaung KuoAffiliation: Instituite of Plant Biology, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a4614
 and Yao-Moan Huang
Yao-Moan Huang Affiliation: Division of Silviculture, Taiwan Forestry Research Institute, Taipei, Taiwan
For correspondence: huangym@tfri.gov.tw
Bio-protocol author page: a4615
,  date: 6/5/2017, 276 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2322.

[Abstract] Seedless vascular plants, including ferns and lycophytes, produce spores to initiate the gametophyte stage and to complete sexual reproduction. Approximately 10% of them are apomictic through the production of genomic unreduced spores. Being able to measure the spore nuclear DNA content is therefore important to infer their reproduction mode. Here ...

DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis

Authors: Jihyeon Yu*
Jihyeon YuAffiliation: School of Biological Sciences, Seoul National University, Seoul, South Korea
Bio-protocol author page: a4643
, Kwangryul Baek*
Kwangryul BaekAffiliation: Department of Life Science, Hanyang University, Seoul, South Korea
Bio-protocol author page: a4644
, EonSeon Jin
EonSeon JinAffiliation: Department of Life Science, Hanyang University, Seoul, South Korea
For correspondence: esjin@hanyang.ac.kr
Bio-protocol author page: a4645
 and Sangsu Bae
Sangsu BaeAffiliation: Department of Chemistry, Hanyang University, Seoul, South Korea
For correspondence: sangsubae@hanyang.ac.kr
Bio-protocol author page: a4646
 (*contributed equally to this work),  date: 6/5/2017, 370 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2352.

[Abstract] We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow covers from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set ...

Single Molecule RNA FISH in Arabidopsis Root Cells

Authors: Susan Duncan
Susan DuncanAffiliation: Earlham Institute, Norwich Research Park, Norwich, United Kingdom
Bio-protocol author page: a4387
, Tjelvar S. G. Olsson
Tjelvar S. G. OlssonAffiliation: John Innes Centre, Norwich Research Park, Norwich, United Kingdom
Bio-protocol author page: a4388
, Matthew Hartley
Matthew HartleyAffiliation: John Innes Centre, Norwich Research Park, Norwich, United Kingdom
Bio-protocol author page: a4389
, Caroline Dean
Caroline DeanAffiliation: John Innes Centre, Norwich Research Park, Norwich, United Kingdom
Bio-protocol author page: a4390
 and Stefanie Rosa
Stefanie RosaAffiliation: John Innes Centre, Norwich Research Park, Norwich, United Kingdom
Present address: Institute of Biochemistry and Biology, Plant Physiology, University of Potsdam, DE-14476 Potsdam-Golm, Germany
For correspondence: srosa@uni-potsdam.de
Bio-protocol author page: a2658
,  date: 4/20/2017, 724 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2240.

[Abstract] Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule ...

Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins

Authors: Rosario Iglesias
Rosario IglesiasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4233
, Lucía Citores
Lucía CitoresAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4234
 and José M. Ferreras
José M. FerrerasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
For correspondence: rosario@bio.uva.es
Bio-protocol author page: a4232
,  date: 3/20/2017, 876 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2180.

[Abstract] Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This ...

Knock-in Blunt Ligation Utilizing CRISPR/Cas9

Authors: Jonathan M. Geisinger
Jonathan M. GeisingerAffiliation: Department of Biology, Stanford University, Stanford, USA
For correspondence: jonmg54@stanford.edu
Bio-protocol author page: a4146
 and Michele P. Calos
Michele P. CalosAffiliation: Department of Genetics, Stanford University, Stanford, USA
Bio-protocol author page: a4147
,  date: 3/5/2017, 834 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2163.

[Abstract] The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in ...

A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

Authors: Johan Vad-Nielsen*
Johan Vad-NielsenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: johanvn@biomed.au.dk
Bio-protocol author page: a3868
, Lin Lin *
Lin Lin Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: lin.lin@biomed.au.dk
Bio-protocol author page: a3869
, Kristopher Torp Jensen
Kristopher Torp JensenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3870
, Anders Lade Nielsen
Anders Lade Nielsen Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3871
 and Yonglun Luo
Yonglun LuoAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3872
 (*contributed equally to this work),  date: 12/5/2016, 1579 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2059.

[Abstract] The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional ...

Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes

Authors: Mariana Edith Tellechea*
Mariana Edith TellecheaAffiliation 1: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari,Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Affiliation 2: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3780
, Javier Garcia-Pardo*
Javier Garcia-PardoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3781
, Juliana Cotabarren
Juliana CotabarrenAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3782
, Daniela Lufrano
Daniela LufranoAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3783
, Laura Bakas
Laura BakasAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3784
, Francesc Xavier Avilés
Francesc Xavier AvilésAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3785
, Walter David Obregon
Walter David ObregonAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3786
, Julia Lorenzo
Julia LorenzoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
For correspondence: julia.lorenzo@uab.es
Bio-protocol author page: a3787
 and Sebastián Tanco
Sebastián TancoAffiliation 1: Medical Biotechnology Center, VIB, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
For correspondence: sebastian.tanco@vib-ugent.be
Bio-protocol author page: a3788
 (*contributed equally to this work),  date: 12/5/2016, 1226 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2032.

[Abstract] Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of new MCP inhibitors ...

Mungbean Yellow Mosaic India Virus (MYMIV)-infection, Small RNA Library Construction and Deep Sequencing for MicroRNA Identification in Vigna mungo

Authors: Anirban Kundu
Anirban KunduAffiliation: Division of Plant Biology, Bose Institute, Kolkata, West Bengal, India
Bio-protocol author page: a2047
, Sujay Paul
Sujay PaulAffiliation: Division of Plant Biology, Bose Institute, Kolkata, West Bengal, India
Bio-protocol author page: a2046
, Amita Pal
Amita PalAffiliation: Division of Plant Biology, Bose Institute, Kolkata, West Bengal, India
For correspondence: amita@jcbose.ac.in
Bio-protocol author page: a2048
 and Genotypic Technology
Genotypic TechnologyAffiliation: Genotypic Technology Private Limited, #2/13, Balaji Complex, Bangalore, India
Bio-protocol author page: a3589
,  date: 10/20/2016, 1347 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1961.

[Abstract] This protocol describes small RNA library preparation from Vigna mungo total RNA followed by deep sequencing and analysis for microRNA identification.​...
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[Bio101] Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well)

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
,  date: 7/5/2011, 15644 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.90.

[Abstract] This is a simple protocol for isolating genomic DNA from fresh plant tissues. DNA from this experiment can be used for all kinds of genetics studies, including genotyping and mapping. This protocol uses Edward’s extraction buffer to isolate DNA....

[Bio101] A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
,  date: 5/20/2011, 11843 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.70.

[Abstract] This method can be used to free and separate the mesophyll cells from Arabidopsis leaves. The protoplasts that are generated in this way can be used for transient expression for protein activity and subcellular localization assays....

Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay)

Authors: Saiprasad Goud Palusa
Saiprasad Goud PalusaAffiliation: Department of Biology, Colorado State University, Fort Collins, USA
Bio-protocol author page: a1022
 and Anireddy S. N. Reddy
Anireddy S. N. ReddyAffiliation: Department of Biology, Colorado State University, Fort Collins, USA
For correspondence: reddy@colostate.edu
Bio-protocol author page: a1023
,  date: 11/20/2013, 10878 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.967.

[Abstract] RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements ...

Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System

Authors: Kabin Xie
Kabin XieAffiliation: Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, State College, USA
Bio-protocol author page: a1624
, Bastian Minkenberg
Bastian MinkenbergAffiliation: Department of Plant Biology, The Pennsylvania State University, University Park, State College, USA
Bio-protocol author page: a1625
 and Yinong Yang
Yinong YangAffiliation: Department of Plant Biology, The Pennsylvania State University, University Park, State College, USA
For correspondence: yuy3@psu.edu
Bio-protocol author page: a1626
,  date: 9/5/2014, 10863 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1225.

[Abstract] RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes ...

[Bio101] DNA Extraction from Dried Plant Tissues Using 96-well format (cTab Method)

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
,  date: 7/5/2011, 10126 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.89.

[Abstract] This high throughput DNA isolation protocol is used to extract DNA of high quality from plant tissues for various genetics studies, like genotyping, and mapping etc. This protocol uses the well-established CTAB extraction procedure, and has been adapted to be used with 96-well plates....

[Bio101] RNA Isolation from Arabidopsis Pollen Grains

Author: Yongxian Lu,  date: 5/5/2011, 9102 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.67.

[Abstract] This purpose of this experiment is to isolate high quality RNAs from pollen grains, which lays the foundation for further studies, like gene expression analysis and cDNA cloning....

Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei

Authors: Laia Armengot
Laia ArmengotAffiliation: Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain
For correspondence: laia.armengot@uab.cat
Bio-protocol author page: a530
 and Jordi Moreno-Romero
Jordi Moreno-RomeroAffiliation: Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain
For correspondence: jordi.moreno@slu.se
Bio-protocol author page: a529
,  date: 4/5/2013, 6508 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.455.

[Abstract] Micrococcal nuclease (MNase) is able to produce double-strand breaks within nucleosome linker regions. The efficiency of MNase digestion depends on the degree of chromatin compaction, being more easily digested the regions of less compacted chromatin. The MNase protocol described here can be used to ...

Whole Genome Bisulfite Sequencing and DNA Methylation Analysis from Plant Tissue

Authors: Daniela Pignatta
Daniela PignattaAffiliation: Whitehead Institute for Biomedical Research, Cambridge, USA
Bio-protocol author page: a2016
, George W. Bell
George W. BellAffiliation: Whitehead Institute for Biomedical Research, Cambridge, USA
Bio-protocol author page: a2017
 and Mary Gehring
Mary GehringAffiliation 1: Whitehead Institute for Biomedical Research, Cambridge, USA
Affiliation 2: Department of Biology, Massachusetts Institute of Technology, Cambridge, USA
For correspondence: mgehring@wi.mit.edu
Bio-protocol author page: a2018
,  date: 2/20/2015, 6168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1407.

[Abstract] This protocol describes whole genome bisulfite-sequencing library preparation from plant tissue and subsequent data analysis. Allele-specific methylation analysis and genome-wide identification of differentially methylated regions are additional features of the analysis procedure....

VIGS Assays

Authors: Haili Zhang
Haili ZhangAffiliation: ChengDu Institute of Biology, Chinese Academy of Sciences, Cheng Du, China
Bio-protocol author page: a1189
 and Yule Liu
Yule LiuAffiliation: School of Life Sciences, Tsinghua University, Beijing, China
For correspondence: yuleliu@mail.tsinghua.edu.cn
Bio-protocol author page: a1190
,  date: 3/5/2014, 5994 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1057.

[Abstract] Virus-induced gene silencing (VIGS) is a powerful method to study gene function in plants. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector so far. This method was originally developed by the Dinesh-Kumar's group (Liu et al., 2002) . Here, we describe a rapid and high efficient ...

Preparation of cDNA Library for dRNA-seq

Authors: Feng Li
Feng LiAffiliation 1: Department of Plant and Microbial Biology, Plant Gene Expression Center, University of California, Berkeley, USA
Affiliation 2: Agricultural Research Services (USDA-ARS), United States Department of Agriculture, Albany, USA
For correspondence: chdlifeng@mail.hzau.edu.cn
Bio-protocol author page: a180
 and Barbara Baker
Barbara BakerAffiliation 1: Department of Plant and Microbial Biology, Plant Gene Expression Center, University of California, Berkeley, USA
Affiliation 2: Agricultural Research Services (USDA-ARS), United States Department of Agriculture, Albany, USA
For correspondence: bbaker@berkeley.edu
Bio-protocol author page: a181
,  date: 12/5/2012, 5451 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.302.

[Abstract] microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide ...
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