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.
Highlights
The past decade has witnessed remarkable advances in genome editing
A series of genome editing tools such as zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and Cas9 have been developed that enable investigators to manipulate virtually any gene in a wide range of cell types and organisms. Both ZFNs and TALENs have almost the same mode of action with their DNA recognition domains interacting with nucleotides and nuclease catalytic domains producing DNA double strand breaks (DSB)
Mutations caused by TALEN or Cas9 are largely predictable as the induced mutant loci were produced at a fixed target sequence
Summary
The past decade has witnessed remarkable advances in genome editing. A series of genome editing tools such as ZFNs, TALENs and Cas9 have been developed that enable investigators to manipulate virtually any gene in a wide range of cell types and organisms. Current assays have their limitations in that they either require specific sequence in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutation from natural nucleotide polymorphism. Our work provides a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.
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