Genome-wide Translocation Analysis in the 266Q Knock-in Mouse Model of SCA7

To determine whether a predisposition to DNA damage exists in SCA7 and how extensive the predilection to DNA damage might be in SCA7, we used LAM-HTGTS, a powerful high throughput next generation sequencing technique developed for monitoring of DNA double-strand break formation. We modified the LAM-HTGTS protocol by utilizing CRISPR-Cas9 to create the double-strand DNA break at a specific site and also added a step with a 5' methyl cytosine modified primer to promote LpnPI endonuclease cleavage of sealed breaks to enrich for translocation events. Our unbiased native chromosome DNA repair experimentation revealed that expression of polyglutamine-expanded ataxin-7 yielded greatly reduced translocations in comparison to normal ataxin-7, which is consistent with retained canonical NHEJ repair, decreased HDR activity, and decreased SSA repair in SCA7 cells, as the classical NHEJ pathway is known to prevent translocation by ligating broken double-strand breaks. Overall design: Examination of translocation events with genome-wide translocation assay in Neuro-2A cells transiently expressing mutant (92Q) or normal (10Q) ataxin-7. Double strand DNA breaks were introduced by co-expression of Cas9 and sgRNAs targeting two selected genomic loci. For the quality control experiment, only the CRISPR-Cas9 plasmid co-expressing site 2 targeted sgRNA or non-targeted sgRNA were used. Three independent transfection replicates per condition were pooled prior sequencing.