Modified crRNA variants for precise targeting of point mutations in human mitochondrial DNA by CRISPR-Cas12a
Abstract
The CRISPR-Cas12a system, primarily used for nuclear genome editing, operates through RNA-guided cleavage of target DNA. Given the high copy number of mitochondrial DNA (mtDNA) in cells and the pathogenic potential of mtDNA mutations, there is substantial interest in developing methods to selectively eliminate mutant mtDNA without affecting wild-type molecules. We suggest using AsCas12a, with its shorter CRISPR RNA (crRNA) and proven ability to be targeted into human mitochondria, to perform selective cleavage of mutant mtDNA. Here, we assess the potential of AsCas12a and various engineered crRNAs to discriminate between wild-type and mutant mtDNA in vitro and in living human cells. By exploring chemically synthesized full and split crRNAs with various modifications, we identified crRNAs capable of targeting specific mutant mtDNA. We demonstrated the strong influence of magnesium concentration on the ability of crRNAs to discriminate point mutations. Finally, we show that AsCas12a-fused cytosine base editor can be addressed to mitochondria and can direct low but significant specific base editing of mtDNA in human cells. Our study suggests AsCas12a as a promising tool for targeted mutant mtDNA elimination or editing.
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BibTeX
@misc{shebanov2026modified,
title = {{Modified crRNA variants for precise targeting of point mutations in human mitochondrial DNA by CRISPR-Cas12a}},
author = {Nikita Shebanov and Anne-Marie Heckel and Natalia Nikitchina and Anna Pavlova and Inna Pyshnaya and Mariya Meschaninova and Ivan Tarassov and Nina Entelis},
howpublished = {HAL},
year = {2026},
month = sep,
doi = {10.64898/2026.09.09.750411},
url = {https://hal.science/hal-05753269v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Modified crRNA variants for precise targeting of point mutations in human mitochondrial DNA by CRISPR-Cas12a
AU - Nikita Shebanov
AU - Anne-Marie Heckel
AU - Natalia Nikitchina
AU - Anna Pavlova
AU - Inna Pyshnaya
AU - Mariya Meschaninova
AU - Ivan Tarassov
AU - Nina Entelis
PY - 2026
DA - 2026/09/09
PB - HAL
DO - 10.64898/2026.09.09.750411
UR - https://hal.science/hal-05753269v1
AB - The CRISPR-Cas12a system, primarily used for nuclear genome editing, operates through RNA-guided cleavage of target DNA. Given the high copy number of mitochondrial DNA (mtDNA) in cells and the pathogenic potential of mtDNA mutations, there is substantial interest in developing methods to selectively eliminate mutant mtDNA without affecting wild-type molecules. We suggest using AsCas12a, with its shorter CRISPR RNA (crRNA) and proven ability to be targeted into human mitochondria, to perform selective cleavage of mutant mtDNA. Here, we assess the potential of AsCas12a and various engineered crRNAs to discriminate between wild-type and mutant mtDNA in vitro and in living human cells. By exploring chemically synthesized full and split crRNAs with various modifications, we identified crRNAs capable of targeting specific mutant mtDNA. We demonstrated the strong influence of magnesium concentration on the ability of crRNAs to discriminate point mutations. Finally, we show that AsCas12a-fused cytosine base editor can be addressed to mitochondria and can direct low but significant specific base editing of mtDNA in human cells. Our study suggests AsCas12a as a promising tool for targeted mutant mtDNA elimination or editing.
ER -