Tandem Repeat Modification during Double-Strand Break Repair Induced by an Engineered TAL Effector Nuclease in Zebrafish Genome

Abstract

Tandem repeats (TRs) are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome in vivo. Transcription activator-like effector nucleases (TALENs) are widely used in recent years in gene targeting for their specific binding to target sequences when engineered in vitro. Here, we show that the repair of a double-strand break (DSB) induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

“…in the single strand annealing pathway for closure of dsDNA breaks might have become active 48 . Interestingly, contraction of the (CGG⋅CCG)n repeat in the FMR1 gene occurred at a low frequency in induced pluripotent stem cells (iPSCs) derived from fragile X-syndrome patients when cleaved upstream by CRISPR/Cas9, 49 and deletion of a (TG⋅CA)70 dinucleotide repeat was induced by TALEN cleavage 129 bp downstream of the repeat in zebrafish 50 . Extensive DNA loss may thus be a common process if simple tandem repeats are exposed to the DNA repair machinery by nearby DSBs.…”

Secondary structure forming sequences drive SD-MMEJ repair of DNA double-strand breaks

“…However, SD-MMEJ could also serve as a beneficial evolutionary mechanism allowing for the creation of novel SNPs and promoting the expansion and diversification of short sequence repeats. Such a mechanism was recently shown to operate at tandem repeats following creation of a DSB using TALENs in zebrafish ( 59 ). Similarly, SD-MMEJ repair could drive the expansion of small repeats in gene promoters, thereby creating a rapid mechanism by which to alter gene expression ( 60 – 62 ).…”

A Mitochondrial Genome of Rhyparochromidae (Hemiptera: Heteroptera) and a Comparative Analysis of Related Mitochondrial Genomes

“…The main source of size variation is the variable number and length of tandem repeats in the control region 15 . Tandem repetition has been widely observed in the mitochondrial control region in animals 16 , and these duplications are thought to play a role in gene transcription or DNA methylation 17 18 . Moreover, tandem repeat units can be added or subtracted by slipped-strand mispairing during replication 19 , and this type of mutation happens at a rate at least 100,000 times higher compared to the rate of simple point mutations 20 .…”