TY - JOUR

T1 - Specific elimination of m.8993T>G mitochondrial haplotype in NARP cybrid cells by CRISPR-Cas9 system

AU - Zakirova, Elvira

AU - Sergeeva, Svetlana

AU - Morozova, Ksenia

AU - Kiseleva, Elena

AU - Tanaka, Masashi

AU - Mazunin, Ilya

AU - Orishchenko, Konstantin

N1 - Specific elimination of m.8993T>G mitochondrial haplotype in NARP cybrid cells by CRISPR-Cas9 system / E. Zakirova, S. Sergeeva, K. Morozova [et al.] // Scientific Reports. – 2026. – DOI 10.1038/s41598-026-49007-y. – EDN LFMVGX. This work was supported by the Ministry of science and higher education of the Russian Federation, grant #FSUS-2024-0018. S.S., K.M. and E.K. were supported by the Ministry of Science and Higher Education of the Russian Federation via the Institute of Cytology and Genetics SB RAS (No. FWNR-2026-0024).

PY - 2026/4/28

Y1 - 2026/4/28

N2 - Mutations in mitochondrial DNA can cause a wide range of neuromuscular and neurodegenerative diseases in humans. The heteroplasmy level, coexistence of both wild-type and mutant mtDNA within a cell, determines the manifestation and severity of disease symptoms. Therefore, the development of strategies to shift heteroplasmy toward wild-type mtDNA is critical for advancing therapies for mitochondrial disorders. Mitochondrial localization of the modified CRISPR-Cas9 system components was analyzed using western blotting, immunocytochemical staining, confocal microscopy, and immunoelectron microscopy. To assess the heteroplasmy shift, cybrid cell lines carrying the clinically relevant m.8993T>G variant associated with Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome were used. The heteroplasmy level was evaluated by RFLP analysis of PCR-amplified mtDNA fragments encompassing the mutation site. We adapted the widely used CRISPR-Cas9 system, originally designed for editing nuclear DNA, to induce a heteroplasmy shift of the pathogenic m.8993T>G point mutation in human cybrid cells. We demonstrated that the modified components of the CRISPR-Cas9 system – the mitoCas9 nuclease fused to the mitochondrial targeting sequence from COX8A, and a single guide RNA containing a mitochondrial import determinant within the tetraloop – are effectively imported into the mitochondrial matrix. Transient transfection of the modified sgRNA into cybrid cells stably expressing mitoCas9 resulted in a detectable shift in heteroplasmy. Mitochondria-targeted CRISPR-SpCas9 system can selectively reduce the mutant m.8993T>G mtDNA load in human cybrid cells, achieving a reproducible shift in heteroplasmy.

AB - Mutations in mitochondrial DNA can cause a wide range of neuromuscular and neurodegenerative diseases in humans. The heteroplasmy level, coexistence of both wild-type and mutant mtDNA within a cell, determines the manifestation and severity of disease symptoms. Therefore, the development of strategies to shift heteroplasmy toward wild-type mtDNA is critical for advancing therapies for mitochondrial disorders. Mitochondrial localization of the modified CRISPR-Cas9 system components was analyzed using western blotting, immunocytochemical staining, confocal microscopy, and immunoelectron microscopy. To assess the heteroplasmy shift, cybrid cell lines carrying the clinically relevant m.8993T>G variant associated with Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome were used. The heteroplasmy level was evaluated by RFLP analysis of PCR-amplified mtDNA fragments encompassing the mutation site. We adapted the widely used CRISPR-Cas9 system, originally designed for editing nuclear DNA, to induce a heteroplasmy shift of the pathogenic m.8993T>G point mutation in human cybrid cells. We demonstrated that the modified components of the CRISPR-Cas9 system – the mitoCas9 nuclease fused to the mitochondrial targeting sequence from COX8A, and a single guide RNA containing a mitochondrial import determinant within the tetraloop – are effectively imported into the mitochondrial matrix. Transient transfection of the modified sgRNA into cybrid cells stably expressing mitoCas9 resulted in a detectable shift in heteroplasmy. Mitochondria-targeted CRISPR-SpCas9 system can selectively reduce the mutant m.8993T>G mtDNA load in human cybrid cells, achieving a reproducible shift in heteroplasmy.

KW - CRISPR-SpCas9

KW - Heteroplasmy shift

KW - Mitochondria

KW - NARP syndrome

KW - m.8993T>G point mutation

UR - https://www.scopus.com/pages/publications/105043042527

UR - https://elibrary.ru/item.asp?id=90181439

UR - https://www.mendeley.com/catalogue/ce58e126-e94f-397d-85f0-2050c8f7afc4/

U2 - 10.1038/s41598-026-49007-y

DO - 10.1038/s41598-026-49007-y

M3 - Article

C2 - 42049921

VL - 16

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 19745

ER -