Sanger sequencing by capillary electrophoresis can be used to determine the efficiency of CRISPR and TALEN-mediated genome editing in primary transformed cultures. Sanger sequencing is an efficient research method to confirm successful genome edits in transformed cultures and to screen secondary clones for successful editing events. Applied Biosystems™ Minor Variant Finder Software can be used to determine the frequency of SNP changes in clones isolated from secondary cultures.

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The simplicity and cost-effectiveness of the workflow, as well as the uncomplicated data analysis, make Sanger sequencing by capillary electrophoresis a valuable part of any genome editing workflow. Sanger sequencing of individual subclones not only facilitates determination of the efficiency of the edit, but also the range of sequence deletions and insertions produced. Moreover, Sanger sequencing provides an easy path to identifying and confirming genomic edits in secondary clones of individual cells.

Overall workflow for CRISPR-mediated genome editing

Establish single-cell clones
run sequence
Characterize successful edits
run sequence

Appropriate assay for your system

Behind the Bench blog post

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

By Swati Kadam, PhD

Genome editing is not a new concept to the scientific community and has been around for decades. However, directing precise sequence changes at desired sites has remained a difficult and tedious challenge for researchers. Limited successes have been achieved with oligonucleotides, small molecules,or self-splicing introns, but the development of site-directed zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) has facilitated sequence-specific manipulations. Nevertheless, difficulties of protein design, synthesis, and testing have slowed adoption of these engineered nucleases for routine use in genome editing experiments. The most recent gene editing technology, the CRISPR-Cas9 system, largely overcomes these difficulties, making it an attractive method for genome editing. It is critical that we adopt a workflow and use technology that can make genome editing efficient by accurately identifying on- and off-target mutations, producing a gene-edited organism that may have fewer or no off-targeted gene side effects. Today, with the aid of gene editing tools, researchers can 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.

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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.

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