TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats

Abstract

Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington's disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.

The role of fork stalling and DNA structures in causing chromosome fragility

“…184 Sae2 is also needed for the repair of a TALEN-induced DSB at a hairpin-forming CTG sequence. 185 Our data at Flex1 led us to propose a new theory for CFS fragility-that the regions are not only prone to breakage but also have difficulty healing after fragility due to neighboring secondary structures. 36 Fragile sites have long been known to be associated with gene amplification through a breakage-fusionbridge cycle.…”

Creating cell and animal models of human disease by genome editing using CRISPR/Cas9

“…Therefore, this process is considered as an applicable technique for producing model systems aiming to identify therapeutic strategies based on genome engineering. [23][24][25][26][27][28][29] The inefficiency of this method lies in the interference of epigenetic changes in the genome with the activity of TALENs. 30 The potential advantage of TALENS over ZFNs is that TALENs are not limited to binding to 9-18-bp target sites.…”

“…So far, TALENs have been used to make purposeful alterations in sequences of interest in the genome of a wide variety of organisms, including humans, rats, worms, zebrafish, pigs and yeast. Therefore, this process is considered as an applicable technique for producing model systems aiming to identify therapeutic strategies based on genome engineering . The inefficiency of this method lies in the interference of epigenetic changes in the genome with the activity of TALENs .…”

Experimental DNA- or RNA-Directed Therapies for Trinucleotide Repeat Disease

“…Like zinc finger nucleases and CRISPR)/CRISPR-Cas9, transcription activator-like effector nucleases (TALENs) are engineered nucleases that have been widely used to generate double-strand DNA breaks (DSBs) to increase the efficiency of standard homologous recombination. Mosbach et al [49] showed that a TALENinduced double-stranded break was very efficient at contracting expanded CTG repeats in yeast. This study showed that, following the TALEN-induced double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction [49].…”

“…Mosbach et al [49] showed that a TALENinduced double-stranded break was very efficient at contracting expanded CTG repeats in yeast. This study showed that, following the TALEN-induced double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction [49]. These results implicate TALENs as a promising strategy for gene therapy of trinucleotide repeat disorders; however, although TALENs have a higher efficiency than zinc finger proteins, it requires huge resources, and delivery into cells is often a challenge [33,34].…”