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

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas9 gene editing relies on single-guide RNA (sgRNA) for target recognition and Cas9 nuclease activity.
• Modifying sgRNA bases can potentially improve stability, functionality, and therapeutic applications.
• Understanding base modifications' impact on Cas9-sgRNA-DNA interactions is crucial for optimizing gene editing.

Purpose of the Study:

• To establish an enzymatic method for generating base-modified RNA constructs.
• To evaluate the functionality of modified sgRNAs in CRISPR-Cas9 cleavage assays.
• To investigate the role of specific base modifications and interactions in Cas9 activity.

Main Methods:

• Enzymatic synthesis of partially and fully base-modified RNA constructs using thienoguanosine (thG) and other analogs.
• Transcriptional efficiency assessment using T7 RNA polymerase variants.
• In vitro CRISPR-Cas9 cleavage assays with modified sgRNAs and PCR products containing modified bases.

Main Results:

• Successfully generated partially and fully modified RNA constructs, including fluorescent thG analogs.
• Modified sgRNAs with thG and thG/5-bromocytosine demonstrated comparable efficacy to natural sgRNAs in CRISPR-Cas9 cleavage.
• N1-Methylpseudouridine and 7-deazapurines were evaluated for their impact on Cas9 activity, highlighting the importance of the purine 7-nitrogen atom.

Conclusions:

• Developed a versatile enzymatic method for producing modified RNA constructs for CRISPR-Cas9 applications.
• Demonstrated the functional equivalence of certain modified sgRNAs to natural sgRNAs, expanding possibilities for RNA-based technologies.
• Provided insights into base-specific interactions critical for Cas9-mediated cleavage, informing future therapeutic development.