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Investigation of DNA double-strand break repair mechanisms in microsatellite regions using the CRISPR/Cas9 system

ABSTRACT: Expansion of the CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntington's disease (HD), spinocerebellar ataxias (SCAs) and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG repeats in yeast models. Understanding this mechanism is the first step to developing a therapeutic strategy based on controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products in the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of mutations. The location of the cleavage site and its sequence affect the outcome of DNA repair. DSBs within CAG repeats result in shortening of the repeats in frame in ~90% of products. The mechanism of this contraction involves MRE11-CTIP and RAD51 activity and extensive DNA end resection reaching ~5000 bp. We demonstrated that a DSB located upstream of CAG repeats induces polymerase theta-mediated end joining, resulting in deletion of the entire CAG tract. Furthermore, using unbiased proteomic analysis, we identified novel factors that may be involved in CAG sequence repair.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell

SUBMITTER: Agata Malinowska

LAB HEAD: Marta Olejniczak

PROVIDER: PXD044960 | Pride | 2024-11-04

REPOSITORIES: Pride

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			Action	DRS
	212224435sle_1-1.raw	Raw
	212224436sle_1-2.raw	Raw
	212224437sle_1-3.raw	Raw
	212224438sle_1-4.raw	Raw
	212224439sle_1-5.raw	Raw

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