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.
Nucleic Acids Res. 2023 Feb 28;51(4):1501-1511. doi: 10.1093/nar/gkac1147.
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.
已经成功建立了一种酶法，能够生成部分碱基修饰的 RNA（以前称为 RZA）构建体，其中所有 G 残基都被同晶荧光噻鸟嘌呤（thG）类似物取代，以及完全修饰的 RZA，其特征是 thG、5-溴胞嘧啶、7-脱氮腺嘌呤和 5-氯尿嘧啶。发现发射完全修饰的 RZA 的转录效率受益于各种 T7 RNA 聚合酶变体的使用。此外，Taq DNA 聚合酶可以与其他三种碱基修饰核苷酸一起将 dthG 掺入 PCR 产物中。值得注意的是，含有 thG 以及 thG 和 5-溴胞嘧啶的所得 RNA 产物在体外 CRISPR-Cas9 切割测定中可与天然 sgRNA 一样有效地发挥作用。还证明了 N1-甲基假尿嘧啶在掺入 sgRNA 时是尿嘧啶的忠实非经典替代物，可指导 Cas9 核酸内切酶切割。7-脱氮嘌呤对 Cas9 的失活表明嘌呤的 7-氮原子在 sgRNA 和 PAM 位点中对于实现有效的 Cas9 切割的重要性。这项研究的其他方面与 sgRNA-蛋白和 PAM-蛋白相互作用的意义有关，这些相互作用在 Cas9-sgRNA-DNA 复合物晶体结构中没有得到强调。这些发现可以扩大 CRISPR-Cas9 和其他基于 RNA 的技术的影响和治疗价值。
