Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2′-fluoro and Locked Nucleic Acid Nucleotides Full article
| Journal |
International Journal of Molecular Sciences
, E-ISSN: 1422-0067 |
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| Output data | Year: 2022, Volume: 23, Number: 21, Article number : 13460, Pages count : DOI: 10.3390/ijms232113460 | ||||
| Tags | CRISPR; crRNA; tracrRNA; sgRNA; gene editing; 2’-modification; LNA; 2’-fluoro RNA; 2’-O-methyl RNA; off-target effect | ||||
| Authors |
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| Affiliations |
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Funding (2)
| 1 | МИНИСТЕРСТВО НАУКИ И ВЫСШЕГО ОБРАЗОВАНИЯ РОССИЙСКОЙ ФЕДЕРАЦИИ | ПФНИ РФ (2021-2030) 0245-2021-0007 |
| 2 | Russian Science Foundation | РНФ №22-14-00294 |
Abstract:
: The genome editing approach using the components of the CRISPR/Cas system has found
wide application in molecular biology, fundamental medicine and genetic engineering. A promising
method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by
modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA,
tracrRNA and sgRNA) containing modified nucleotides (2’-O-methyl, 2’-fluoro, LNA—locked nucleic
acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion
and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide
RNAs. The replacement of ribonucleotides with 2’-fluoro modified or LNA nucleotides increased the
lifetime of the crRNAs, while other types of modification did not change their nuclease resistance.
Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise,
the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy.
The kinetic constant of DNA cleavage was higher for the system with 2’-fluoro modified crRNA. The
2’-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage
Cite:
Sakovina L.
, Vokhtantsev I.P.
, Vorobyeva M.A.
, Vorobyev P.E.
, Novopashina D.S.
Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2′-fluoro and Locked Nucleic Acid Nucleotides
International Journal of Molecular Sciences. 2022. V.23. N21. 13460 . DOI: 10.3390/ijms232113460 WOS Scopus РИНЦ OpenAlex
Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2′-fluoro and Locked Nucleic Acid Nucleotides
International Journal of Molecular Sciences. 2022. V.23. N21. 13460 . DOI: 10.3390/ijms232113460 WOS Scopus РИНЦ OpenAlex
Dates:
| Published print: | Nov 3, 2022 |
Identifiers:
| Web of science: | WOS:000883472200001 |
| Scopus: | 2-s2.0-85141623655 |
| Elibrary: | 57643539 |
| OpenAlex: | W4308331310 |