CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.

CRISPR-Cas Systems DNA Gene Editing / methods Nucleic Acids RNA / chemistry Fluorescence Guanosine / chemistry

Journal

Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011

Informations de publication

Date de publication:
28 02 2023
Historique:
accepted: 17 11 2022
revised: 09 11 2022
received: 13 10 2022
pubmed: 8 1 2023
medline: 4 3 2023
entrez: 7 1 2023
Statut: ppublish

Résumé

An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies. With the advent of CRISPR-Cas9 gene editing, we now have to hand a simple two-component system amendable to silencing and knock-in editing effectively any gene. Yet we must not forget that the implications of immunotoxicity along with the poor stability and specificity of canonical nucleic acids hold enormous challenges for in vivo applications, especially in gene therapy. Our study endorses the feasibility of the enzymatic approach to incorporate nucleobase modifications into the CRISPR-Cas9 system unveiling the tolerance of Cas9 to N1-methylpseudouridine (m1Ψ)- and emissive thienoguanosine (thG)-modified sgRNA as well as thus far uncharted structural requirements for ensuring proper PAM recognition.

Autres résumés

Type: plain-language-summary (eng)
With the advent of CRISPR-Cas9 gene editing, we now have to hand a simple two-component system amendable to silencing and knock-in editing effectively any gene. Yet we must not forget that the implications of immunotoxicity along with the poor stability and specificity of canonical nucleic acids hold enormous challenges for in vivo applications, especially in gene therapy. Our study endorses the feasibility of the enzymatic approach to incorporate nucleobase modifications into the CRISPR-Cas9 system unveiling the tolerance of Cas9 to N1-methylpseudouridine (m1Ψ)- and emissive thienoguanosine (thG)-modified sgRNA as well as thus far uncharted structural requirements for ensuring proper PAM recognition.

Identifiants

DOI: 10.1093/nar/gkac1147 PMID: 36611237 PMC: PMC9976875
pubmed: 36611237
pii: 6967240
doi: 10.1093/nar/gkac1147
pmc: PMC9976875
doi:

Substances chimiques

DNA 9007-49-2
Nucleic Acids 0
RNA 63231-63-0
thienoguanosine 0
Guanosine 12133JR80S

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

1501-1511

Informations de copyright

© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Auteurs

Hui Yang (H)

KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, Box 1041, 3000 Leuven, Belgium.

Elena Eremeeva (E)

KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, Box 1041, 3000 Leuven, Belgium.
Queensland University of Technology, Centre for Agriculture and the Bioeconomy, Molecular Engineering Group, George Street 2, 4000 Brisbane, Queensland, Australia.

Mikhail Abramov (M)

KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, Box 1041, 3000 Leuven, Belgium.

Maarten Jacquemyn (M)

KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Herestraat 49, Box 1043, 3000 Leuven, Belgium.

Elisabetta Groaz (E)

KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, Box 1041, 3000 Leuven, Belgium.
University of Padova, Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 35131 Padova, Italy.

Dirk Daelemans (D)

KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Herestraat 49, Box 1043, 3000 Leuven, Belgium.

Piet Herdewijn (P)

KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, Box 1041, 3000 Leuven, Belgium.
ORCID: 0000-0003-3589-8503

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Classifications MeSH

Recherchemedicale.com › Phénomènes génétiques › Régulation de l'expression des gènes › Épigenèse génétique › Extinction de l'expression des gènes › Systèmes CRISPR-Cas › CRISPR-Cas9 recognition of enzymatically...
Recherchemedicale.com › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ADN › CRISPR-Cas9 recognition of enzymatically...
Recherchemedicale.com › Techniques d'investigation › Techniques génétiques › Génie génétique › Édition de gène › CRISPR-Cas9 recognition of enzymatically...
Recherchemedicale.com › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › CRISPR-Cas9 recognition of enzymatically...
Recherchemedicale.com › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › CRISPR-Cas9 recognition of enzymatically...
Recherchemedicale.com › Phénomènes physiques › Phénomènes optiques › Fluorescence › CRISPR-Cas9 recognition of enzymatically...
Recherchemedicale.com › Acides nucléiques, nucléotides et nucléosides › Nucléosides › Ribonucléosides › Guanosine › CRISPR-Cas9 recognition of enzymatically...