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

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 fo...

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Detalles Bibliográficos
Autores principales: VanLeuven, Ariel J, Park, Sungdae, Menke, Douglas B, Lauderdale, James D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9580339/
https://www.ncbi.nlm.nih.gov/pubmed/29939088
http://dx.doi.org/10.2144/btn-2018-0012
Descripción
Sumario: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.