ArticleNucleic acids research2023
CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.
Article in Nucleic acids research, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- Distinct Phage-Encoded Enzymes for Substitution of Deoxythymidine by Deoxyuridine in Phage Genomes.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Article
- Coloring target and off-target effects of genetically modified nucleases by blue & white colony assays.Biotechnology letters · 2025Article
- The pucke.rs toolkit to facilitate sampling the conformational space of biomolecular monomers.Journal of cheminformatics · 2025Article
- The positional and numerical effect ofRSC advances · 2024Article
- Harnessing noncanonical crRNA for highly efficient genome editing.Nature communications · 2024Article
- Structural insights into the morpholino nucleic acid/RNA duplex using the new XNA builder Ducque in a molecular modeling pipeline.Nucleic acids research · 2024Article
- Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Influence of N1-Methylpseudouridine in Guide RNAs on CRISPR/Cas9 Activity.International journal of molecular sciences · 2023Article
- Antiretrovirals to CCR5 CRISPR/Cas9 gene editing - A paradigm shift chasing an HIV cure.Clinical immunology (Orlando, Fla.) · 2023Review
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Authors and funding
7 authors.
Funding
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Abstract
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.
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