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PLoS One. 2014 Jun 5;9(6):e98282. doi: 10.1371/journal.pone.0098282. eCollection 2014.

A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.

Author information

1
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital and College of Life Science, Sichuan University, Chengdu, Sichuan, People's Republic of China.

Abstract

Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.

PMID:
24901507
PMCID:
PMC4046980
DOI:
10.1371/journal.pone.0098282
[Indexed for MEDLINE]
Free PMC Article

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