A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish. (2018)

• Record Type: Journal Article
• Authors: VanLeuven, Ariel J; Park, Sungdae; Menke, Douglas B; Lauderdale, James D
• Is Part Of: Biotechniques. Volume 64:Number 6(2018)
• Page Start: 275
• Page End: 278
• Publication Date: 2018
• Subjects: Danio rerio -- genotyping -- polymerase chain reaction (PCR); Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/btn-2018-0012
• Languages: English
• ISSNs: 0736-6205

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.