Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions

PLoS Genetics
Valentine MosbachGuy-Franck Richard

Abstract

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversi...Continue Reading

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Citations

Feb 14, 2021·Journal of Huntington's Disease·Vanessa C Wheeler, Vincent Dion
Dec 7, 2020·Current Opinion in Genetics & Development·Rebecca E Brown, Catherine H Freudenreich
Sep 1, 2021·Current Opinion in Genetics & Development·Erica J Polleys, Catherine H Freudenreich

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Datasets Mentioned

BETA
PRJEB16068

Methods Mentioned

BETA
PCR

Software Mentioned

IndelRealigner
IGV
GATK
R package
Mpileup
CRISPOR
Varscan2
Image Lab
FastQC
BWA

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