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.

Because the repair process is not completely efficient or accurate in genome editing experiments, fragment analysis and/or Sanger sequencing by capillary electrophoresis (CE) can be used to determine the efficiency of CRISPR-mediated genome editing in primary transformed cell cultures, and Sanger sequencing can be used to determine successful editing events in secondary clones.

Sanger sequencing is the gold standard technology for confirmation of genome editing due to its accuracy, cost-effective and simple workflow, long read lengths, and straightforward data analysis, while fragment analysis can quickly and inexpensively detect insertions and deletions with single–base pair resolution.

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

Overall workflow for CRISPR-mediated genome editing

Thermo Fisher Scientific has integrated all the tools necessary for genome editing and downstream analysis.


Step 1

Design and synthesize gRNA
identify variants

Step 2

Transfect cells
choose primers

Step 3

Determine frequency of successful edits
prepare

Step 4

Establish single-cell clones
run sequence

Step 5

Screen clones for edits
analyze data

Step 6

Characterize successful edits
run sequence
  • Appropriate assay for your system

CRISPR-Cas9 genome editing analysis with SeqStudio and SeqStudio Flex genetic analyzers

The features of the SeqStudio and SeqStudio Flex genetic analyzers facilitate Sanger sequencing and fragment analysis in the same run which enables them to fit well within a genome editing workflow. Pair with the Applied Biosystem SeqScreener Gene Edit Confirmation app to help validate your CRISPR editing efficiently and with confidence.

SeqScreener Gene Edit Confirmation App

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.

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Featured resources

Genetic analysis tools for genome editing workflows

Each method for evaluating genome editing efficiency has its own strengths—learn more about them, and our complete workflow solutions for genome editing experiments.


Application note: Using Sanger sequencing to facilitate CRISPR- and TALEN-mediated genome editing workflows

In this application note, a genome editing workflow is described that includes efficiency determination and identification by Sanger sequencing with data analysis through Minor Variant Finder Software. The results of a CRISPR-mediated edit are presented, but the approach can also be applied to ZFN- and TALEN-mediated editing workflows.


Blog post: CRISPR-Cas9 genome editing guide—finessing the technique and breaking new ground

This blog discusses the history of various gene editing techniques from TALEN to CRISPR-Cas9 systems, and the challenges and advancements for accurately identifying on- and off-target mutations. The authors explore the possibilities of gene editing tools to quickly generate model organisms that can be used to study human diseases, test efficacy of various drugs, and even create genetically modified organisms during times of an epidemic, as has been done for the Aedes aegypti mosquito, a vector for the Zika virus.

Read the full blog post ›

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