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

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 ((th)G) analogs, as well as fully modified RZA featuring (th)G, 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, d(th)G could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing (th)G as well as (th)G together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N-1-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. Lay 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 N-1-methylpseudouridine (m1 psi)- and emissive thienoguanosine ((th)G)-modified sgRNA as well as thus far uncharted structural requirements for ensuring proper PAM recognition.