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

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-Methyl

SUBMITTER: Yang H 

PROVIDER: S-EPMC9976875 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature