High Efficiency In Vivo Genome Engineering with a Simplified 15-RVD GoldyTALEN Design
- Authors
- Ma, A.C., Lee, H.B., Clark, K.J., and Ekker, S.C.
- ID
- ZDB-PUB-130709-2
- Date
- 2013
- Source
- PLoS One 8(5): e65259 (Journal)
- Registered Authors
- Clark, Karl, Ekker, Stephen C., Lee, Han B.
- Keywords
- none
- MeSH Terms
-
- Animals
- Genome/genetics*
- INDEL Mutation*
- Mutagenesis, Site-Directed/methods*
- Polymerase Chain Reaction
- Polymorphism, Restriction Fragment Length
- Reproducibility of Results
- Zebrafish/genetics*
- Zebrafish Proteins/genetics
- PubMed
- 23734242 Full text @ PLoS One
Transcription activator-like effector nucleases (TALENs) enable genome engineering in cell culture and many organisms. Recently, the GoldyTALEN scaffold was shown to readily introduce mutations in zebrafish (Danio rerio) and livestock through non-homologous end joining (NHEJ) and homology-directed repair (HDR). To deploy the GoldyTALEN system for high-throughput mutagenesis in model organisms, a simple design with high efficacy is desirable. We tested the in vivo efficacy of a simplified 15-RVD GoldyTALEN design (spacer between 13–20 bp and T nucleotide preceding each TALEN binding site) in zebrafish. All 14 tested TALEN pairs (100%) introduced small insertions and deletions at somatic efficacy ranging from 24 to 86%, and mutations were inheritable at high frequencies (18–100%). By co-injecting two GoldyTALEN pairs, inheritable deletions of a large genomic fragment up to 18 kb were successfully introduced at two different loci. In conclusion, these high efficiency 15-RVD GoldyTALENs are useful for high-throughput mutagenesis in diverse application including hypothesis testing from basic science to precision medicine.