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

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.

SUBMITTER: VanLeuven AJ

PROVIDER: S-EPMC9580339 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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

VanLeuven Ariel J AJ Park Sungdae S Menke Douglas B DB Lauderdale James D JD

BioTechniques 20180601 6

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 app ...[more]

PMID: 29939088

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