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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 ...

Multiplex Gene Editing via CRISPR/Cas9 System in Sheep

Authors: Yiyuan Niu
Yiyuan NiuAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4803
, Yi Ding
Yi DingAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4804
, Xiaolong Wang
Xiaolong WangAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4805
 and Yulin Chen
Yulin ChenAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
For correspondence: chenyulin@nwafu.edu.cn
Bio-protocol author page: a4802
,  date: 7/5/2017, 133 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2385.

[Abstract] Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified ...

Dense sgRNA Library Construction Using a Molecular Chipper Approach

Authors: Jijun Cheng
Jijun ChengAffiliation 1: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 2: Yale Stem Cell Center, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA
For correspondence: j.cheng@yale.edu
Bio-protocol author page: a4740
, Wen Pan
Wen PanAffiliation 1: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 2: Yale Stem Cell Center, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA
Bio-protocol author page: a3289
 and Jun Lu
Jun LuAffiliation 1: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 2: Yale Stem Cell Center, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 3: Yale Cooperative Center of Excellence in Hematology, Yale University, New Haven, Connecticut, USA
Affiliation 4: Yale Center for RNA Science and Medicine, Yale University, New Haven, Connecticut, USA
For correspondence: jun.lu@yale.edu
Bio-protocol author page: a4741
,  date: 6/20/2017, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2373.

[Abstract] Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. ...

In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair

Authors: Melike Çağlayan
Melike ÇağlayanAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
Bio-protocol author page: a1849
 and Samuel H. Wilson
Samuel H. WilsonAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
For correspondence: wilson5@niehs.nih.gov
Bio-protocol author page: a1850
,  date: 6/20/2017, 232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2341.

[Abstract] We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β and DNA ligase ...

Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity

Authors: Trung M. Hoang
Trung M. HoangAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
Bio-protocol author page: a4648
, Tatiana Kent
Tatiana KentAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
Bio-protocol author page: a4649
 and Richard T. Pomerantz
Richard T. PomerantzAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
For correspondence: richard.pomerantz@temple.edu
Bio-protocol author page: a4650
,  date: 6/20/2017, 175 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2330.

[Abstract] DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human ...

Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes

Authors: David J. Nolan
David J. NolanAffiliation 1: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Affiliation 2: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4654
, Susanna L. Lamers
Susanna L. LamersAffiliation: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Bio-protocol author page: a4655
, Rebecca Rose
Rebecca RoseAffiliation: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Bio-protocol author page: a4656
, James J. Dollar
James J. DollarAffiliation 1: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Affiliation 2: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4657
, Marco Salemi
Marco SalemiAffiliation: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4658
 and Michael S. McGrath
Michael S. McGrathAffiliation 1: Departments of Laboratory Medicine, Pathology, and Medicine, University of California at San Francisco, San Francisco, California, USA
Affiliation 2: The AIDS and Cancer Specimen Resource, University of California at San Francisco, San Francisco, California, USA
For correspondence: MMcGrath@php.ucsf.edu
Bio-protocol author page: a4659
,  date: 6/20/2017, 180 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2334.

[Abstract] The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al., 2016a and 2016b; Rose ...

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 ...

Tagged Highly Degenerate Primer (THDP)-PCR for Community Analysis of Methane- and Ammonia-oxidizing Bacteria Based on Copper-containing Membrane-bound Monooxygenases (CuMMO)

Authors: Jian-Gong Wang
Jian-Gong WangAffiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China
Bio-protocol author page: a4717
, Fei Xia
Fei XiaAffiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China
Bio-protocol author page: a4718
, Jemaneh Zeleke
Jemaneh ZelekeAffiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China
Bio-protocol author page: a4719
, Bin Zou
Bin ZouAffiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China
Bio-protocol author page: a4720
 and Zhe-Xue Quan
Zhe-Xue QuanAffiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China
For correspondence: quanzx@fudan.edu.cn
Bio-protocol author page: a4721
,  date: 6/20/2017, 158 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2354.

[Abstract] We describe a two-step PCR strategy using tagged highly degenerate primer (THDP-PCR) targeting copper-containing membrane-bound monooxygenases (CuMMO) genes for community analysis of methane- or ammonia-oxidizing bacteria. This strategy consists of a primary CuMMO gene-specific PCR followed by a secondary PCR with a tag as a single primer. This strategy ...

Targeted Nucleotide Substitution in Mammalian Cell by Target-AID

Authors: Takayuki Arazoe
Takayuki ArazoeAffiliation: Graduate school of Science, Technology and Innovation, Kobe University, Hyogo, Japan
Bio-protocol author page: a4638
, Keiji Nishida
Keiji NishidaAffiliation: Graduate school of Science, Technology and Innovation, Kobe University, Hyogo, Japan
For correspondence: keiji_nishida@people.kobe-u.ac.jp
Bio-protocol author page: a4639
 and Akihiko Kondo
Akihiko KondoAffiliation: Graduate school of Science, Technology and Innovation, Kobe University, Hyogo, Japan
For correspondence: akondo@kobe-u.ac.jp
Bio-protocol author page: a1574
,  date: 6/5/2017, 421 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2339.

[Abstract] Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed ...

Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs

Authors: Vincent Loubiere
Vincent LoubiereAffiliation 1: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
Affiliation 2: University of Montpellier, 163 Rue Auguste Broussonnet, 34090 Montpellier, France
Bio-protocol author page: a4619
, Anna Delest
Anna DelestAffiliation: Centre de Recherche en Cancérologie de Lyon, INSERM U1052, 151 cours Albert Thomas, 69003 Lyon, France
Bio-protocol author page: a4620
, Bernd Schuettengruber
Bernd SchuettengruberAffiliation: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
Bio-protocol author page: a4621
, Anne-Marie Martinez
Anne-Marie MartinezAffiliation 1: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
Affiliation 2: University of Montpellier, 163 Rue Auguste Broussonnet, 34090 Montpellier, France
For correspondence: anne-marie.martinez@igh.cnrs.fr
Bio-protocol author page: a4622
 and Giacomo Cavalli
Giacomo CavalliAffiliation: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
For correspondence: giacomo.cavalli@igh.cnrs.fr
Bio-protocol author page: a4623
,  date: 6/5/2017, 285 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2327.

[Abstract] Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples ...
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[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fanglian09@gmail.com
Bio-protocol author page: a9
,  date: 2/5/2011, 93431 views, 32 Q&A
DOI: https://doi.org/10.21769/BioProtoc.30.

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits....

[Bio101] E. coli Genomic DNA Extraction Updates
The author made some updates (highlighted in blue) to the protocol on 09/12/2016.

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a9
,  date: 7/20/2011, 86431 views, 47 Q&A
DOI: https://doi.org/10.21769/BioProtoc.97.

[Abstract] This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits....

[Bio101] DNA Molecular Weight Calculation

Author: Fanglian He,  date: 3/20/2011, 37630 views, 7 Q&A
DOI: https://doi.org/10.21769/BioProtoc.46.

[Abstract] This method is to roughly estimate DNA molecular weight. One of its applications is to calculate the ratio of vector to insert in a ligation reaction (please see Standard DNA Cloning protocol).
Anhydrous molecular weight of each nucleotide is (see reference 1):
A= 313.21
T= 304.2
C= 289.18
G=329.21
For rough ...





[Bio101] Calcium Phosphate Transfection of Eukaryotic Cells

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
,  date: 2/5/2012, 28859 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.86.

[Abstract] Transfection of DNA into cells is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA ...

C2C12 Myoblasts

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
,  date: 5/20/2012, 28470 views, 11 Q&A
DOI: https://doi.org/10.21769/BioProtoc.172.

[Abstract] C2C12 myoblasts are commonly used in biomedical laboratories as an in vitro system to study muscle development and differentiation. This protocol explains the basic procedures of culture, transfection and differentiation of C2C12 myoblast cells....

[Bio101] A General EMSA (Gel-shift) Protocol

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
,  date: 2/5/2011, 26229 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.24.

[Abstract] An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe ...

[Bio101] Standard DNA Cloning

Author: Fanglian He,  date: 4/5/2011, 26041 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.52.

[Abstract] This protocol describes general cloning steps from preparation of both vector and insert DNA to the ligation reaction....

[Bio101] Lentivirus and Retrovirus Transfection

Author: Yanlin Huang,  date: 3/5/2011, 20326 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.38.

[Abstract] 293T and Pheonix cells grow in DMEM + 10%FBS. If you are transfecting other cells, you can use whatever medium those cells normally grow in and change to DMEM + 10%FBS on the day of the transfection. You can change back to “normal” medium 24 hours post-transfection. Calcium Phosphate transfection of ...

[Bio101] Lentivirus Infection

Author: Nabila Aboulaich,  date: 2/20/2011, 18650 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.37.

[Abstract] ...

[Bio101] Preparation of Taq DNA Polymerase

Author: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
,  date: 10/5/2011, 16720 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.136.

[Abstract] ...
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