A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish

Ariel J VanLeuven, Sungdae Park, Douglas B Menke, James D Lauderdale

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

The introduction of CRISPR-Cas9 technology for targeted mutagenesis has revolutionized reverse genetics and made genome editing a realistic option in many model organisms. One of the difficulties with this technique is screening for mutations in large numbers of samples. Many screening approaches for identifying CRISPR-Cas9 mutants have been published; however, in practice these methods are time consuming, expensive, or often yield false positives. This report describes a PCR-based screening approach using non-denaturing PAGE. This approach does not depend on the formation of heteroduplexes and reliably detects changes as small as 1 base-pair (bp) in nucleic acid length at the target site. This approach can be used to identify novel mutations and is also useful as a routine genotyping method.

Original languageEnglish (US)
Pages (from-to)275-278
Number of pages4
JournalBioTechniques
Volume64
Issue number6
DOIs
StatePublished - Jun 2018

Keywords

  • Animals
  • CRISPR-Cas Systems/genetics
  • DNA/analysis
  • Genotyping Techniques/methods
  • Mutation/genetics
  • Native Polyacrylamide Gel Electrophoresis/methods
  • Polymerase Chain Reaction/methods
  • Zebrafish/genetics

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