Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Chemistry

Background:

• CRISPR-Cas technology has transformed genome editing, increasing demand for synthetic guide RNAs.
• The synthesis of chemically modified single-guide RNAs (sgRNAs) exceeding 100 nucleotides is a significant challenge.
• Current methods for producing long sgRNAs are inefficient and limit broader applications.

Purpose of the Study:

• To develop an efficient method for synthesizing long single-guide RNAs (sgRNAs).
• To overcome the bottleneck in producing chemically modified sgRNAs for CRISPR-Cas applications.
• To demonstrate the utility of the novel sgRNA synthesis method in human cells.

Main Methods:

• A tetrazine ligation strategy was employed for sgRNA synthesis.
• A tetrazine moiety was attached to the 3'-end of crRNA and a norbornene moiety to the 5'-end of tracrRNA.
• Ligation was performed under mild conditions to form the complete sgRNA molecule.

Main Results:

• The tetrazine ligation method successfully produced chemically modified sgRNAs.
• Tetrazine-ligated sgRNAs demonstrated efficient genome editing at reporter and endogenous gene loci in human cells.
• Optimization of the linker structure was found to be crucial for achieving high-efficiency editing.

Conclusions:

• Tetrazine ligation offers a robust and efficient method for preparing long, chemically modified sgRNAs.
• This approach addresses a critical limitation in CRISPR-Cas technology, facilitating advanced research and therapeutic development.
• Further structural optimization of the ligation linker can enhance genome editing efficiency.