CRISPR Cas9 Gene Editing Validation Using Sanger Sequencing

CRISPR mediated genome editing techniques greatly facilitate targeted genome modifications, such as deletions or insertions, in complex organisms. In recent years, the CRISPR-Cas9 system has been established as the editing technique of choice due to ease of use, cost effectiveness, and the high-efficacy of desired targeted edits.

The Applied Biosystems SeqScreener Gene Edit Confirmation App (SGC) is a free and user-friendly software to determine the range and frequency of mutations generated in your CRISPR-Cas9 experiments. Combined with Applied Biosystems genetic analysis instruments and the Sanger sequencing workflow, you can screen and validate your gene-editing results quickly and easily.

Applied Biosystems genetic analysis instruments and reagents provide:

  • Easy and unambiguous identification of single nucleotide polymorphisms (SNPs), sequence deletions, and insertions produced in a cellular population during gene editing
  • Compatible web-based software, SeqScreener Gene Edit Confirmation app, to calculate gRNA efficiency and consistency, predict experiment outcomes, and validate clones for research use
  
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Intuitive design—The free and easy-to-use software enables quick identification of successful gene knock-ins and knockouts. The visual workflow integrates two modes: screening the gRNA efficiency in a pool of cells and screening an array of edited cell clones.


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Fast and accurate—The robust algorithm calculates the editing frequencies and grades the editing outcome for each sample in a matter of seconds. Results of the SeqScreener app correlate strongly
with NGS-based results in a wide range of editing percentages.


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Convenient—Available on the cloud via the Thermo Fisher Connect Platform, analysis can be done virtually anywhere with no software to download or install. With the batch processing capabilities, up to 2,400 files can be uploaded and analysed all at once.

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About the SeqScreener Gene Edit Confirmation App on Thermo Fisher Connect

The SeqScreener Gene Edit Confirmation App (SGC) is an application available through Thermo Fisher Connect that can be used to determine the spectrum and frequency of targeted mutations generated in a pool of cells by genome editing tools such as CRISPR-Cas9. Use the SGC app to screen and validate gene editing results obtained using Capillary Electrophoresis (CE) sequencing technology.

The SGC app includes the following features:

  • An algorithm that accurately reconstructs the spectrum of Indels from the sequence traces and generates a report
  • A report output that identifies the detected Indels and their frequencies
  • Detection of designed mutations generated by homologous recombination using a donor template
  • Compatibility with both knock-in and knock-out experiments
  • Batch import capability
  • Pool screening and clonal screening analyses

Learn more about the app


Featured resources

Genetic analysis tools for genome editing workflows

 

Invitrogen TrueDesign Genome Editor

The Invitrogen TrueDesign Genome Editor is a free online tool that enables scientists of all experience levels to easily design, select, and order reagents for accurate and successful gene editing experiments. Its intuitive point-and-type interface makes it easy to find and successfully edit a gene in a streamlined three-step process: search, edit, and design.

The TrueDesign tool supports five types of edits—gene knockout, fluorescent tagging, insertion, deletion, and SNP replacement—across five species. For each design, it compiles a list of the materials you will need for a successful edit, with the convenience of ordering them from one source and the confidence that they will work together. Our research shows that improved design and delivery of gRNA, Cas9 nuclease, and donor DNA can contribute to enhanced CRISPR/Cas9-mediated genome editing.

Tip: We also offer pre-designed synthetic and lentiviral guide RNAs for straightforward knockout of your human or mouse target gene, plus easy online ordering of any custom synthetic sgRNA design.

Capabilities

Within this simple step-by-step tool, you can:

  • Generate a complete gene editing design using CRISPR-Cas9 or TAL effector nucleases in just minutes
  • Generate an SNP in your target gene
  • Achieve highly effective knockout with insertion of stop codons
  • Review your comprehensive design and protocols and export them to an Excel spreadsheet
  • Delete, insert, or replace up to 30 bases in any human, mouse, rat, zebrafish, or roundworm gene using CRISPR-Cas9 or TALEN technology
  • Add a GFP or RFP tag to a target gene without the need for cloning
  • Design all necessary tools for precise tagging, SNP, amino acid, or knockout changes
  • Order all materials with one click from a single source with confidence that they will work together seamlessly 

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Webinar:

Simplified Online Tools for CRISPR Cas9 Gene Editing Design and Confirmation

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Style Sheet for Global Design System

For Research Use Only. Not for use in diagnostic procedures.