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

Hui Yang; Elena Eremeeva; Mikhail Abramov; Maarten Jacquemyn; Elisabetta Groaz; Dirk Daelemans; Piet Herdewijn

doi:10.1093/nar/gkac1147

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

Nucleic Acids Res. 2023 Feb 28;51(4):1501-1511. doi: 10.1093/nar/gkac1147.

Authors

Hui Yang¹, Elena Eremeeva^{1

2}, Mikhail Abramov¹, Maarten Jacquemyn³, Elisabetta Groaz^{1

4}, Dirk Daelemans³, Piet Herdewijn¹

Affiliations

¹ KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, Box 1041, 3000 Leuven, Belgium.
² Queensland University of Technology, Centre for Agriculture and the Bioeconomy, Molecular Engineering Group, George Street 2, 4000 Brisbane, Queensland, Australia.
³ 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.
⁴ University of Padova, Department of Pharmaceutical and Pharmacological Sciences, Via Marzolo 5, 35131 Padova, Italy.

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.

Plain language summary

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.

Publication types

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

MeSH terms

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

Substances

DNA
Nucleic Acids
RNA
thienoguanosine
Guanosine