Journal Article

CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair

Olejniczak, Marta, Smielowska, Marianna Iga, Sledzinski, Pawel, Nowaczyk, Mateusz

2024

Overview

Background The expansion of 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/CTG repeats in yeast models. Understanding this mechanism is the first step in developing a therapeutic strategy based on the controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products at the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of sequences. Results The location of the cleavage site and the surrounding sequence influence 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 DNA end resection. 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 proteomic analysis, we identified novel factors that may be involved in CAG sequence repair. Conclusions Our study provides new insights into the complex mechanisms of CRISPR/Cas9-induced shortening of CAG repeats in human cells.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Analysis / Biomedical and Life Sciences / Cell culture / Cells / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / DNA / DNA Breaks, Double-Stranded / DNA damage / DNA Repair / Double-strand break repair / Dystrophy / Gene deletion / Gene sequencing / Genes / Genetic aspects / Genetic engineering / Genetic polymorphisms / Genome editing / Genomes / Genomics / Health aspects / Homology / Humans / Huntingtin / Huntingtin Protein - genetics / Huntingtin Protein - metabolism / Huntington's disease / Huntingtons disease / Kinases / Life Sciences / MRE11 protein / Myotonic dystrophy / Nervous system diseases / Neurodegenerative diseases / Nucleotide sequence / Polyglutamine / Proteins / Proteomics / Recombinational DNA Repair / Research Article / Short tandem repeats / Spinocerebellar ataxia / Surgery / TMEJ / Trinucleotide repeat diseases / Trinucleotide Repeat Expansion - genetics / Trinucleotide repeats / Trinucleotide Repeats - genetics / Yeast