Abstract
Gene editing allows to make a variety of targeted changes in genome, which can potentially be used to treat hereditary human diseases. Despite numerous studies in this area, effectiveness of gene editing methods for correcting mutations is still low, so these methods are not allowed in routine practice. It has been shown that rational design of genome editing components can significantly increase efficiency of mutation correction. In this work, we propose design of single-stranded oligodeoxyribonucleotides (ssODNs) to increase efficiency of gene editing. Using a model system to repair knocked out EGFP that is integrated into the genome of HEK293T cell culture, we have shown that only a small part of ssODN (about 20 nucleotides: from the 15th nucleotide at 3′-end to the 4th nucleotide at 5′-end), a donor molecule for repairing double-stranded DNA breaks, is integrated into the site of the break. Based on the obtained data, it is possible to rationally approach the design of ssODNs to correct mutations using CRISPR-Cas9 method for the development of gene therapy for hereditary human diseases.
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Abbreviations
- Cas:
-
CRISPR-associated protein
- CRISPR:
-
clustered regularly interspaced short palindromic repeats
- DSB:
-
double-strand break
- NHEJ:
-
non-homologous end joining
- sgRNA:
-
spacer of guide RNA
- ssODN:
-
single-stranded oligodeoxyribonucleotide
- SSTR:
-
single strand template repair
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Acknowledgments
The authors are grateful to Ph.D. N. G. Gurskaya from the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, for assistance in obtaining the HEK293T-GFPmut cell culture.
Funding
This work was financially supported by the State Budget Project of the Ministry of Science and Higher Education of the Russian Federation for Research Centre for Medical Genetics. Creation of the HEK293T-GFPmut cell culture was funded by the Russian Foundation for Basic Research, grant No. 19-29-04044.
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The authors declare no conflicts of interest in financial or any other sphere. This article does not contain any studies with human participants or animals performed by any of the authors.
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Volodina, O.V., Anuchina, A.A., Zainitdinova, M.I. et al. Rational Design of ssODN to Correct Mutations by Gene Editing. Biochemistry Moscow 87, 464–471 (2022). https://doi.org/10.1134/S0006297922050078
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DOI: https://doi.org/10.1134/S0006297922050078