CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

doi:10.1007/s11427-020-1819-8

. 2021 Sep;64(9):1463-1472.

doi: 10.1007/s11427-020-1819-8. Epub 2021 Jan 6.

CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

Bang Wang¹, Xiujuan Lv¹, Yufei Wang¹, Zhibo Wang¹, Qi Liu², Bin Lu³, Yong Liu¹, Feng Gu⁴

Affiliations

¹ School of Ophthalmology and Optometry, Eye Hospital, State Key Laboratory of Ophthalmology, Optometry and Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, 325027, China.
² Department of Endocrinology & Metabolism, Shanghai Tenth People's Hospital, Tongji University, Shanghai, 200092, China.
³ Attardi Institute of Mitochondrial Biomedicine, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.
⁴ School of Ophthalmology and Optometry, Eye Hospital, State Key Laboratory of Ophthalmology, Optometry and Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, 325027, China. gufenguw@gmail.com.

PMID: 33420919
DOI: 10.1007/s11427-020-1819-8

CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

Bang Wang et al. Sci China Life Sci. 2021 Sep.

. 2021 Sep;64(9):1463-1472.

doi: 10.1007/s11427-020-1819-8. Epub 2021 Jan 6.

Authors

Bang Wang¹, Xiujuan Lv¹, Yufei Wang¹, Zhibo Wang¹, Qi Liu², Bin Lu³, Yong Liu¹, Feng Gu⁴

Affiliations

¹ School of Ophthalmology and Optometry, Eye Hospital, State Key Laboratory of Ophthalmology, Optometry and Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, 325027, China.
² Department of Endocrinology & Metabolism, Shanghai Tenth People's Hospital, Tongji University, Shanghai, 200092, China.
³ Attardi Institute of Mitochondrial Biomedicine, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.
⁴ School of Ophthalmology and Optometry, Eye Hospital, State Key Laboratory of Ophthalmology, Optometry and Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, 325027, China. gufenguw@gmail.com.

PMID: 33420919
DOI: 10.1007/s11427-020-1819-8

Abstract

Genetic manipulation of mitochondrial DNA (mtDNA) could be harnessed for deciphering the gene function of mitochondria; it also acts as a promising approach for the therapeutic correction of pathogenic mutation in mtDNA. However, there is still a lack of direct evidence showing the edited mutagenesis within human mtDNA by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9). Here, using engineered CRISPR/Cas9, we observed numerous insertion/deletion (InDel) events at several mtDNA microhomologous regions, which were triggered specifically by double-strand break (DSB) lesions within mtDNA. InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor, iniparib, demonstrating the evidence of rewiring DSB repair status to manipulate mtDNA using CRISPR/Cas9. These findings would provide novel insights into mtDNA mutagenesis and mitochondrial gene therapy for diseases involving pathogenic mtDNA.

Keywords: CRSIPR/Cas9; genome editing; microhomologous region; mtDNA.

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References

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[1] Antón, Z., Mullally, G., Ford, H.C., van der Kamp, M.W., Szczelkun, M.D., and Lane, J.D. (2020). Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors. J Cell Sci 133, jcs248468. - PubMed - DOI

[2] Antón, Z., Mullally, G., Ford, H.C., van der Kamp, M.W., Szczelkun, M.D., and Lane, J.D. (2020). Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors. J Cell Sci 133, jcs248468. - PubMed - DOI

[3] Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa, A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson, C., Newby, G.A., Raguram, A., et al. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157. - PubMed - PMC - DOI

[4] Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa, A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson, C., Newby, G.A., Raguram, A., et al. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157. - PubMed - PMC - DOI

[5] Bacman, S.R., Kauppila, J.H.K., Pereira, C.V., Nissanka, N., Miranda, M., Pinto, M., Williams, S.L., Larsson, N.G., Stewart, J.B., and Moraes, C. T. (2018). MitoTALEN reduces mutant mtDNA load and restores tRN^Ala levels in a mouse model of heteroplasmic mtDNA mutation. Nat Med 24, 1696–1700. - PubMed - PMC - DOI

[6] Bacman, S.R., Kauppila, J.H.K., Pereira, C.V., Nissanka, N., Miranda, M., Pinto, M., Williams, S.L., Larsson, N.G., Stewart, J.B., and Moraes, C. T. (2018). MitoTALEN reduces mutant mtDNA load and restores tRN^Ala levels in a mouse model of heteroplasmic mtDNA mutation. Nat Med 24, 1696–1700. - PubMed - PMC - DOI

[7] Bacman, S.R., Williams, S.L., and Moraes, C.T. (2009). Intra- and intermolecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res 37, 4218–4226. - PubMed - PMC - DOI

[8] Bacman, S.R., Williams, S.L., and Moraes, C.T. (2009). Intra- and intermolecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res 37, 4218–4226. - PubMed - PMC - DOI

[9] Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. - PubMed - PMC - DOI

[10] Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. - PubMed - PMC - DOI

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CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

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CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

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