CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair
Abstract 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 mech...
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2024-12-01
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| Online Access: | https://doi.org/10.1186/s12915-024-02079-6 |
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| author | Pawel Sledzinski Mateusz Nowaczyk Marianna Iga Smielowska Marta Olejniczak |
| author_facet | Pawel Sledzinski Mateusz Nowaczyk Marianna Iga Smielowska Marta Olejniczak |
| author_sort | Pawel Sledzinski |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-00267d9cfd6b453ebb4ede3893e848d3 |
| institution | Kabale University |
| issn | 1741-7007 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biology |
| spelling | doaj-art-00267d9cfd6b453ebb4ede3893e848d32024-12-08T12:46:08ZengBMCBMC Biology1741-70072024-12-0122112110.1186/s12915-024-02079-6CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repairPawel Sledzinski0Mateusz Nowaczyk1Marianna Iga Smielowska2Marta Olejniczak3Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of SciencesDepartment of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of SciencesDepartment of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of SciencesDepartment of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of SciencesAbstract 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.https://doi.org/10.1186/s12915-024-02079-6DNA repairGenome editingShort tandem repeatsTMEJ |
| spellingShingle | Pawel Sledzinski Mateusz Nowaczyk Marianna Iga Smielowska Marta Olejniczak CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair BMC Biology DNA repair Genome editing Short tandem repeats TMEJ |
| title | CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair |
| title_full | CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair |
| title_fullStr | CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair |
| title_full_unstemmed | CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair |
| title_short | CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair |
| title_sort | crispr cas9 induced double strand breaks in the huntingtin locus lead to cag repeat contraction through dna end resection and homology mediated repair |
| topic | DNA repair Genome editing Short tandem repeats TMEJ |
| url | https://doi.org/10.1186/s12915-024-02079-6 |
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