We use cookies to improve your browsing experience and provide meaningful content. Read our cookie policy. Accept

Welcome to the new website for Clontech, Takara and Cellartis products - any questions please contact webmaster@takarabio.com

Close
  •  Customer Login
  • Register
  •  View Cart (0)
  •  Customer Login
  • Register
  •  View Cart (0)

  • Products
  • Learning centers
  • Services & Support
  • About
  • Areas of interest

Close

  • ‹ Back to Gene editing in hiPS cells
  • Tagging an endogenous gene with AcGFP1 in hiPS cells
  • Tagging an endogenous gene with a myc tag in hiPS cells
  • Generating clonal hiPS cell lines deficient in CD81
  • Introducing a tyrosinemia-related SNP in hiPS cells
  • Inserting an expression cassette into the AAVS1 locus in hiPS cells
  • Editing hiPS cells using electroporation
  • Editing hiPS cells using gesicle technology
  • Single-cell cloning of hiPS cells
Gene editing service contact us Contact us for gene editing services
Home › Learning centers › Stem cell research › Technical notes › Gene editing in hiPS cells › Generating clonal hiPS cell lines deficient in CD81

Stem cell technical notes

  • Pluripotent stem cells
    • Using the DEF-CS system to culture human iPS cells
    • Comparison of the Cellartis DEF-CS system with other vendors' human iPS cell culture systems
    • Reprogramming PBMCs
    • Reprogramming fibroblasts
  • Gene editing in hiPS cells
    • Tagging an endogenous gene with AcGFP1 in hiPS cells
    • Tagging an endogenous gene with a myc tag in hiPS cells
    • Generating clonal hiPS cell lines deficient in CD81
    • Introducing a tyrosinemia-related SNP in hiPS cells
    • Inserting an expression cassette into the AAVS1 locus in hiPS cells
    • Editing hiPS cells using electroporation
    • Editing hiPS cells using gesicle technology
    • Single-cell cloning of hiPS cells
  • Beta cells
    • Beta cells for disease modeling
  • Cardiomyocytes
    • Making engineered heart tissue with cardiomyocytes
  • Hepatocytes
    • Hepatocytes for disease modeling
    • Hepatocytes for drug metabolism studies
    • Long-term human primary hepatocyte culture
    • iPS cell to hepatocyte differentiation system
  • Neural stem cells
    • RHB-A neural stem cell medium
New products
Need help?
Gene editing service contact us Contact us for gene editing services
Case Study

Knocking out an endogenous gene (CD81) in hiPS cells

The combination of footprint-free gene editing using CRISPR/Cas9 and hiPS cells allows for the generation of disease models that are essential for advancing our understanding of disease mechanisms and the development of novel therapeutics. One of the most powerful applications of genome editing is the capacity to specifically knock out a certain gene through the introduction of indels via the nonhomologous end joining (NHEJ) repair pathway, which leads to frameshift mutations and/or premature stop codons. Knocking out a gene of interest enables the study of gene function; for example, by knocking out a disease-causing gene, one can gain a better understanding of underlying disease mechanisms as well as possible therapeutic interventions. Here, we describe our workflow for the targeted knockout of the endogenous gene CD81 in hiPS cells.

Experimental workflow sgRNA design Analysis of edited population Characterization of clonal cell lines Conclusions

Experimental workflow  

Our workflow for knocking out the CD81 gene begins with hiPS cells cultured in our Cellartis DEF-CS 500 Culture System, which provides a homogeneous, undifferentiated starting population. To edit the hiPS cells, we delivered the Cas9-sgRNA complex in the form of ribonucleoprotein (RNP) in order to decrease off-target effects and for footprint-free genome editing. Since CD81 is a protein in the extracellular membrane, FACS sorting can be used to select CD81-knockout cells from the edited population, and then single cells can be seeded and expanded to generate clonal cell lines.

Workflow for generating CD81 knock out cell lines

sgRNA design  

A good experimental design is crucial for efficient and successful gene editing. In order to knock out the CD81 gene, we chose an sgRNA targeting the first exon. The sgRNA was in vitro transcribed and had an optimized scaffold sequence to enhance binding to Cas9 and form a more stable complex. After incubating the sgRNA with Cas9 protein, the Cas9-sgRNA RNP complex was introduced to hiPS cells via electroporation.

Analysis of edited population  

A week after the gene editing experiment, cells were analyzed using flow cytometry. Since CD81 is a membrane protein, staining the cells with an anti-CD81 antibody labeled with FITC gave a direct and accurate percentage of the cells deficient in CD81, which is equivalent to successful gene editing by CRISPR/Cas9. Almost 90% of the cells had CD81 knocked out.

CD81-expressing cells were detected by flow cytometry

Flow cytometry data from edited and control hiPSCs

Characterization of clonal cell lines  

The next phase of the establishment of the edited clonal cell lines was to sort the subpopulation of cells knocked out for CD81 and single-cell isolate them by flow cytometry. Single cells were expanded to clonal cell lines using our DEF-CS single-cell cloning system that ensures a single-cell survival rate higher than 60%. Several clonal cell lines were assessed for knockout of CD81 as well as pluripotency via flow cytometry. All clones exhibited a loss of CD81 expression. Pluripotency was maintained in all of the edited clonal lines, as evidenced by the expression of the three pluripotency markers at levels comparable to those in the parental line.

Checking for pluripotency markers

CRISPR/Cas9-mediated editing and single-cell cloning can be harsh on hiPS cells, and it can force selective pressures that favor unintended mutations that affect the karyotype. To confirm karyotype stability, we examined several of the expanded clonal lines that were deficient in CD81. All lines were found to have normal, stable karyotypes. These data show that our genome editing and single-cell cloning protocols effectively edit and expand hiPS cell clones without introducing karyotypic abnormalities.

Karyotype analysis of edited clones

Simultaneously, we characterized the specific insertions or deletions (indels) created during the CRISPR/Cas9 editing process in the clonal cell lines using our protocol for indel identification, followed by Sanger sequencing. Because Cas9-induced double-strand breaks are mainly repaired via the error-prone NHEJ repair pathway, clonal cell lines can exhibit different indels in each allele, and these indels can vary between cell lines. The results showed that each clone had a unique set of indels that led to the knockout of CD81.

Sequencing analysis of edited clones

Conclusions  

We have developed a complete workflow for the knockout of an endogenous gene. Our workflow consists of footprint-free CRISPR/Cas9-mediated editing, single-cell cloning of the edited population, and characterization of expanded clonal cell lines to identify positive clones. The edited hiPS cells maintain pluripotency and have normal, stable karyotypes, which are important for downstream applications such as directed differentiation.

Let our team of experts create custom edited clonal cell lines for your research. Contact us today so we can get started on your project.


Gene editing services

Gene editing

Generate knockouts and knockins in your human iPS cells

With our expertise in CRISPR/Cas9 technology and human pluripotent stem cell culture, we will efficiently edit your iPS cells.

Learn more

Takara Bio USA, Inc.
United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2018 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.

Takara Bio USA, Inc. (TBUSA, formerly known as Clontech Laboratories, Inc.) provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, TBUSA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

Support
  • Contact us
  • Technical support
  • Customer service
  • Shipping & delivery
  • Sales
  • Feedback
Products
  • New products
  • Special offers
  • Instrument & reagent services
  • Corporate development
Learning centers
  • NGS
  • Gene function
  • Stem cell research
  • Protein research
  • PCR
  • Cloning
  • Nucleic acid purification
About
  • Our brands
  • Careers
  • Events
  • Blog
  • Need help?
  • Announcements
  • Quality and compliance
  • That's Good Science!
Facebook Twitter  LinkedIn

©2019 Takara Bio Inc. All Rights Reserved.

Region - North America Privacy Policy Terms and Conditions Terms of Use

Top



  • Automation systems
  • SmartChip Real-Time PCR System, chips, and reagents
  • SMARTer Apollo system
  • SMARTer ICELL8 systems
  • Next-generation sequencing
  • Single-cell RNA- and DNA-seq
  • RNA-seq
  • DNA-seq
  • Immune profiling
  • Real-time PCR
  • Real-time PCR kits
  • RNA extraction and analysis for real-time qPCR
  • Reverse transcription prior to qPCR
  • Stem cell research
  • Media and supplements
  • Stem cells and stem cell-derived cells
  • Human iPS cell gene editing systems
  • Nucleic acid purification
  • Plasmid purification kits
  • Genomic DNA purification kits
  • DNA cleanup kits
  • RNA purification kits
  • cDNA synthesis
  • cDNA synthesis kits
  • Reverse transcriptases
  • RACE kits
  • Purified cDNA & genomic DNA
  • Purified total RNA and mRNA
  • PCR
  • High-yield PCR
  • High-fidelity PCR
  • GC rich PCR
  • Lyophilized master mixes
  • Cloning
  • In-Fusion Cloning
  • Competent cells
  • Ligation kits
  • Restriction enzymes
  • Legacy cloning products
  • Nucleic acid extraction
  • Cell biology assays
  • Exosome isolation (cell culture)
  • Reporter systems
  • Apoptosis detection kits
  • Epigenetics
  • Signal transduction
  • Gene function
  • Gene editing
  • Fluorescent proteins
  • Viral transduction
  • Tet-inducible expression systems
  • Transfection reagents
  • Protein research
  • Purification products
  • Mass spectrometry reagents
  • Two-hybrid and one-hybrid systems
  • Antibodies and ELISAs
  • Primary antibodies and ELISAs by research area
  • Fluorescent protein antibodies
  • New products
  • Special offers
  • CRISPR-Cas9 promotion
  • RT-qPCR bundle promotion
  • Lenti-X special offers
Create a web account with us

Log in to enjoy additional benefits

Why sign up for an account?

An account with takarabio.com entitles you to extra features such as:

•  Creating and saving shopping carts
•  Keeping a list of your products of interest
•  Saving all of your favorite pages on the site
•  Accessing restricted content

Create an account to get started

  • Automation systems
  • SmartChip Real-Time PCR System introduction
  • SMARTer Apollo library prep system introduction
  • SMARTer ICELL8 introduction
  • Next-generation sequencing
  • Selection guide
  • Product line overview
  • Technical notes
  • FAQs and tips
  • Webinars
  • Posters
  • Real-time PCR
  • Product finder
  • Reaction size guidelines for qPCR
  • Real-time PCR products brochure
  • Real-time PCR tutorial videos
  • Overview
  • Technical notes
  • FAQs
  • Stem cell research
  • Application protocols
  • Technical notes
  • Webinars
  • Videos
  • Citations
  • Nucleic acid purification
  • Product finder
  • Plasmid purification
  • Genomic DNA purification
  • DNA/RNA cleanup and extraction
  • RNA purification
  • Hard-to-lyse samples
  • cDNA synthesis
  • PCR
  • Citations
  • Selection guides
  • PCR enzyme brochure
  • Technical notes
  • PCR FAQs
  • Cloning
  • In-Fusion Cloning tools
  • In-Fusion Cloning guide
  • In‑Fusion Cloning FAQs
  • In‑Fusion Cloning tips
  • Choosing a seamless cloning method
  • Seamless cloning primer design
  • Cell biology assays
  • Gene function
  • Gene editing
  • Viral transduction
  • Inducible systems
  • Transfection reagents
  • Fluorescent proteins
  • Protein research
  • Capturem rapid purification technology
  • His-tag purification
  • Antibody purification
  • Phosphoprotein and glycoprotein purification
  • Mass spectrometry digestion reagents
  • Matchmaker Gold yeast two-hybrid systems
  • Antibodies and ELISA
Capturem Trypsin for a rapid, efficient mass spectometry workflow at room temperature.

Speed up your mass spec workflow

Capturem Trypsin provides rapid, efficient, and complete digestion of protein samples, allowing an uninterrupted mass spectometry workflow at room temperature for downstream protein analysis. This product utilizes our novel Capturem technology in a spin column format with membrane-immobilized trypsin. Capturem Trypsin Columns may be used to completely digest protein samples in less than a minute with digestion efficiencies (protein coverage) comparable to or better than those obtained using in-solution trypsin digestion.

Capturem trypsin technology

  • Customer service
  • Sales
  • Shipping & delivery
  • Technical support
  • Trademarks
  • License statements
  • Vector information
  • Vector document overview
  • Vector document finder
  • Website FAQs
  • Feedback
  • Business development
  • OEM, custom, and supply-chain solutions
  • In licensing
  • Out licensing
  • Submit a licensing request
  • Instrument & reagent services
  • Instrument services
  • Cell and gene therapy manufacturing services
  • Stem cell services
  • Takara Bio affiliates & distributors
  • United States and Canada
  • China
  • Japan
  • Korea
  • Europe
  • India
  • Affiliates & distributors, by country
Takara Bio's award-winning GMP-compliant manufacturing facility in Kusatsu, Shiga, Japan.

Partner with Takara Bio!

Takara Bio is proud to offer GMP-grade manufacturing capabilities at our award-winning facility in Kusatsu, Shiga, Japan.

Learn more

  • Announcements
  • Events
  • Calendar
  • Conferences
  • Careers
  • Quality statement
  • Our brands
  • Takara
  • Clontech
  • Cellartis
  • That's Good Science!
  • Season one
  • Season two
  • Season three
  • BioView blog
  • Need help?

Mapping the brain, one cell type at a time

Learn about pioneering efforts to map the mammalian brain using single-cell transcriptomics.

Watch video

  • Cancer research
  • Sample prep from FFPE tissue
  • Sample prep from plasma
  • Cancer biomarker discovery
  • Cancer biomarker quantification
  • Single cancer cell analysis
  • Cancer genomics and epigenomics
  • HLA typing in cancer
  • Gene editing for cancer therapy/drug discovery
  • Immunotherapy research
  • T-cell therapy
  • Antibody therapeutics
  • T-cell receptor profiling
  • TBI initiatives in cancer therapy

 Customer Login
 View Cart (0)
  • Home
  • Products
  • Learning centers
  • Services & Support
  • Areas of interest
  • Feedback
  • About
  •  Customer Login
  • Register
  •  View Cart (0)

Takara Bio USA, Inc. (TBUSA, formerly known as Clontech Laboratories, Inc.) provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, TBUSA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

Clontech, TaKaRa, cellartis

  • Products
  • Automation systems
  • Next-generation sequencing
  • Gene function
  • Stem cell research
  • Protein research
  • PCR
  • Cloning
  • Nucleic acid purification
  • Antibodies and ELISA
  • Cell biology assays
  • Real-time PCR
  • cDNA synthesis
  • Automation systems
  • SmartChip Real-Time PCR System, chips, and reagents
  • SMARTer Apollo system
  • SMARTer ICELL8 systems
  • Next-generation sequencing
  • Single-cell RNA- and DNA-seq
  • RNA-seq
  • DNA-seq
  • Immune profiling
  • Epigenetics and small RNA sequencing
  • Sequencing accessories
  • Gene function
  • Gene editing
  • Fluorescent proteins
  • Viral transduction
  • Tet-inducible expression systems
  • ProteoTuner protein control systems
  • iDimerize inducible protein interaction systems
  • Transfection reagents
  • Mammalian expression plasmids
  • Stem cell research
  • Media and supplements
  • Stem cells and stem cell-derived cells
  • Human iPS cell gene editing systems
  • Accessories
  • Protein research
  • Purification products
  • Expression vectors & systems
  • Antibodies and immunoprecipitation
  • Mass spectrometry reagents
  • Protein sequencing
  • Glycobiology
  • Two-hybrid and one-hybrid systems
  • SDS-PAGE & western blotting
  • Accessory enzymes
  • PCR
  • Standard PCR
  • High-yield PCR
  • High-fidelity PCR
  • Fast PCR
  • Long-range PCR
  • GC rich PCR
  • Direct PCR
  • Lyophilized master mixes
  • Commercial-use products
  • Molecular diagnostic products
  • GMP-grade products
  • Application-specific PCR
  • Other PCR-related products
  • PCR thermal cyclers
  • Cloning
  • In-Fusion Cloning
  • Competent cells
  • Ligation kits
  • Mutagenesis kits
  • Ligation enzymes
  • Restriction enzymes
  • Modifying enzymes
  • Legacy cloning products
  • X-Gal and IPTG
  • Linkers, primers, and cloning vectors
  • Agarose gel electrophoresis
  • Nucleic acid extraction
  • Nucleic acid purification
  • Plasmid purification kits
  • Genomic DNA purification kits
  • DNA cleanup kits
  • RNA purification kits
  • RNA cleanup kits
  • Viral DNA and RNA purification kits
  • Accessories and components
  • Antibodies and ELISA
  • Primary antibodies and ELISAs by research area
  • Secondary antibodies
  • Antibody and ELISA accessories
  • Fluorescent protein antibodies
  • Cell biology assays
  • Exosome isolation (cell culture)
  • Reporter systems
  • Apoptosis detection kits
  • Epigenetics
  • Cell biology reagents
  • RNA interference
  • Cell-culture accessories
  • Signal transduction
  • Real-time PCR
  • Real-time PCR kits
  • RNA extraction and analysis for real-time qPCR
  • Reverse transcription prior to qPCR
  • Real-time PCR primer sets
  • References and standards for qPCR
  • Application-specific qPCR
  • cDNA synthesis
  • cDNA synthesis kits
  • Reverse transcriptases
  • RACE kits
  • Purified cDNA & genomic DNA
  • Purified total RNA and mRNA
  • cDNA synthesis accessories
  • Learning centers
  • Automation systems
  • Next-generation sequencing
  • Gene function
  • Stem cell research
  • Protein research
  • PCR
  • Cloning
  • Nucleic acid purification
  • Antibodies and ELISA
  • Cell biology assays
  • Real-time PCR
  • cDNA synthesis
  • Automation systems
  • SmartChip Real-Time PCR System introduction
  • SMARTer Apollo library prep system introduction
  • SMARTer ICELL8 introduction
  • Next-generation sequencing
  • Selection guide
  • Product line overview
  • Technical notes
  • Featured kits
  • Technology and application overviews
  • FAQs and tips
  • DNA-seq protocols
  • Webinars
  • Citations
  • Posters
  • Gene function
  • Gene editing
  • Viral transduction
  • Inducible systems
  • Transfection reagents
  • Fluorescent proteins
  • Stem cell research
  • Application protocols
  • Technical notes
  • Posters
  • Webinars
  • Videos
  • FAQs
  • Citations
  • Selection guides
  • Overview
  • Protein research
  • Capturem rapid purification technology
  • His-tag purification
  • Antibody purification
  • Phosphoprotein and glycoprotein purification
  • Other tag purification
  • Mass spectrometry digestion reagents
  • Matchmaker Gold yeast two-hybrid systems
  • Expression systems
  • PCR
  • Citations
  • Selection guides
  • PCR enzyme brochure
  • Technical notes
  • PCR FAQs
  • Go green with lyophilized enzymes
  • LA PCR technology
  • Cloning
  • In-Fusion Cloning tools
  • In-Fusion Cloning guide
  • In‑Fusion Cloning FAQs
  • In‑Fusion Cloning tips
  • Choosing a seamless cloning method
  • Seamless cloning primer design
  • In-Fusion Cloning applications collection
  • Efficient cloning for sgRNA/Cas9 plasmids
  • In-Fusion Cloning tech notes
  • In-Fusion Cloning webinars
  • In-Fusion Cloning citations
  • EcoDry reagents and sustainability
  • Mutagenesis with In-Fusion Cloning
  • Efficient multiple-fragment cloning
  • Sign up to stay updated
  • Traditional molecular cloning
  • Nucleic acid purification
  • Product finder
  • Plasmid purification
  • Genomic DNA purification
  • DNA/RNA cleanup and extraction
  • RNA purification
  • Parallel DNA, RNA & protein
  • Hard-to-lyse samples
  • Antibodies and ELISA
  • Leucine rich repeat-containing protein (LRG)
  • Oncogene research focus
  • mTOR in aging and cancer
  • Osteocalcin focus
  • Cell biology assays
  • Cell viability kits
  • Improved exosome isolation
  • Mir-X microRNA quantification
  • Real-time PCR
  • Product finder
  • Reaction size guidelines for qPCR
  • Real-time PCR products brochure
  • Real-time PCR tutorial videos
  • Overview
  • Technical notes
  • FAQs
  • cDNA synthesis
  • Premium total and poly A+ RNA
  • SMARTer RACE 5'/3' Kit—advances in SMARTer PCR cDNA synthesis
  • Cloning antibody variable regions
  • Services & Support
  • Technical support
  • Shipping & delivery
  • Customer service
  • Sales
  • Website FAQs
  • Vector information
  • Instrument & reagent services
  • Corporate development
  • Takara Bio affiliates & distributors
  • License statements
  • Trademarks
  • Vector information
  • Vector document overview
  • Vector document finder
  • Instrument & reagent services
  • Instrument services
  • Cell and gene therapy manufacturing services
  • Stem cell services
  • Corporate development
  • OEM, custom, and supply-chain solutions
  • In licensing
  • Out licensing
  • Submit a licensing request
  • Takara Bio affiliates & distributors
  • United States and Canada
  • China
  • Japan
  • Korea
  • Europe
  • India
  • Affiliates & distributors, by country
  • Areas of interest
  • Cancer research
  • Immunotherapy research
  • Cancer research
  • Sample prep from FFPE tissue
  • Sample prep from plasma
  • Cancer biomarker discovery
  • Cancer biomarker quantification
  • Single cancer cell analysis
  • Cancer genomics and epigenomics
  • HLA typing in cancer
  • Gene editing for cancer therapy/drug discovery
  • Immunotherapy research
  • T-cell therapy
  • Antibody therapeutics
  • T-cell receptor profiling
  • TBI initiatives in cancer therapy
  • About
  • Manufacturing DSS Takara Bio India
  • Careers
  • Quality and compliance
  • Need help?
  • Our brands
  • Our history
  • Announcements
  • Our partners
  • BioView blog
  • That's Good Science!
  • Special offers
  • New products
  • Events
  • Our brands
  • Takara
  • Clontech
  • Cellartis
  • That's Good Science!
  • Season one
  • Season two
  • Season three
  • Special offers
  • CRISPR-Cas9 promotion
  • RT-qPCR bundle promotion
  • Lenti-X special offers
  • End of the Year Promo (DKK)
  • End of the Year Promo (EU)
  • End of the Year Promo (SEK)
  • End of the Year Promo (CHF)
  • End of the Year Promo (GB)
  • Events
  • Calendar
  • Conferences
  • Products
  • Learning centers
  • Services & Support
  • About
  • Areas of interest