Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides

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Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides. / Sakovina, Lubov; Vokhtantsev, Ivan; Vorobyeva, Mariya et al.

In: International Journal of Molecular Sciences, Vol. 23, No. 21, 13460, 03.11.2022.

Research output: Contribution to journal › Article › peer-review

BibTeX

@article{3ac59dc0c89c42f8a4f111bb19285d3a,

title = "Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides",

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.",

keywords = "2{\textquoteright}-fluoro RNA, 2{\textquoteright}-modification, 2{\textquoteright}-O-methyl RNA, CRISPR, crRNA, gene editing, LNA, off-target effect, sgRNA, tracrRNA",

author = "Lubov Sakovina and Ivan Vokhtantsev and Mariya Vorobyeva and Pavel Vorobyev and Darya Novopashina",

note = "Publisher Copyright: {\textcopyright} 2022 by the authors.",

year = "2022",

month = nov,

day = "3",

doi = "10.3390/ijms232113460",

language = "English",

volume = "23",

journal = "International Journal of Molecular Sciences",

issn = "1661-6596",

publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",

number = "21",

}

RIS

TY - JOUR

T1 - Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides

AU - Sakovina, Lubov

AU - Vokhtantsev, Ivan

AU - Vorobyeva, Mariya

AU - Vorobyev, Pavel

AU - Novopashina, Darya

PY - 2022/11/3

Y1 - 2022/11/3

N2 - 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.

AB - 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.

KW - 2’-fluoro RNA

KW - 2’-modification

KW - 2’-O-methyl RNA

KW - CRISPR

KW - crRNA

KW - gene editing

KW - LNA

KW - off-target effect

KW - sgRNA

KW - tracrRNA

UR - http://www.scopus.com/inward/record.url?scp=85141623655&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/02566e86-62f7-3599-bd4e-cf5f1b489dc7/

U2 - 10.3390/ijms232113460

DO - 10.3390/ijms232113460

M3 - Article

C2 - 36362256

AN - SCOPUS:85141623655

VL - 23

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 21

M1 - 13460

ER -

ID: 39330720