High Efficiency In Vivo Genome Engineering with a Simplified 15-RVD GoldyTALEN Design

Abstract

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.

A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish

“…Several platforms have been established to assemble the highly repetitive TALE repeats, including the PCR-based Golden Gate assembly [8,9,23], plasmid-based Golden Gate assembly [24][25][26][27], fast ligation-based automatable solid-phase high-throughput assembly [28] and ligation-independent cloning assembly [29]. Among these platforms, the plasmid-based Golden Gate assembly has been proven to be an easy, fast and effective method which has been adopted by most laboratories.…”

TALEN Knockout of the PSIP1 Gene in Human Cells: Analyses of HIV-1 Replication and Allosteric Integrase Inhibitor Mechanism

“…Error-prone repair by the cellular nonhomologous end-joining (NHEJ) pathway generates gene-disrupting indels at the target site. Use of two TALEN pairs can produce full deletion of the intervening chromosome DNA (47)(48)(49)(50).…”

An optimized TALEN application for mutagenesis and screening inDrosophila melanogaster

“…The DNA-binding domain we adopted for TALEN design contains modular nucleotide recognition units repeated 14.5 times for optimal DNA recognition and cleavage efficiencies. 18 Each modular unit consists of 34 amino acid residues, the twelfth and thirteenth of which are highly variable (repeat-variable diresidues; RVDs) and are responsible for the one-to-one binding between a modular DNA binding unit and a nucleotide in the DNA molecule. 16,17,19 The type IIS endonuclease, FokI, is fused to the C-terminal end of the DNA-binding domain of TALEs, [20][21][22] as was first accomplished in ZFNs.…”

“…29 However, even with these successful applications, the rates of somatic and germline mutations induced by any given TALEN pair varied widely. 3,18,30,31 The varying rates of mutagenesis may come from intrinsic properties of the TALEN proteins, epigenetic environments of genomic loci, extraneous experimental conditions, or other unknown causes. Currently, detecting an efficient TALEN pair usually depends on sophisticated molecular assays such as restriction fragment length polymorphism (RFLP) and The white gene locus on X chromosome and design of white TALEN pair 1 (w1 TALEN) and pair 2 (w2 TALEN).…”