"Split-and-Click" sgRNA.

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• CRISPR-Cas9 gene editing relies on single guide RNA (sgRNA) for directing Cas9 endonuclease activity.
• Current methods for sgRNA generation, such as enzymatic reactions, are time-consuming and limit chemical modifications.
• Solid-phase RNA synthesis offers an alternative but struggles with producing highly pure, full-length sgRNAs.

Purpose of the Study:

• To develop an efficient and versatile method for synthesizing single guide RNAs (sgRNAs).
• To overcome limitations of current sgRNA production, including time, scalability, and chemical modification incorporation.
• To enable rapid access to custom sgRNA libraries for enhanced CRISPR applications.

Main Methods:

• A "split-and-click" chemical ligation strategy was employed to synthesize sgRNAs.
• The sgRNA was divided into two components: a DNA-targeting 20-mer RNA and a Cas9-binding 79-mer RNA.
• Stringent purification protocols were applied to the DNA-targeting 20-mer component.

Main Results:

• The "split-and-click" approach successfully generated biologically active sgRNAs.
• This method allows for stringent purification of the crucial DNA-targeting RNA component.
• It significantly reduces the synthesis effort for the constant Cas9-binding RNA, enabling rapid library generation.

Conclusions:

• The "split-and-click" method provides a streamlined and efficient route to custom sgRNA synthesis.
• This approach facilitates the incorporation of chemical modifications, expanding CRISPR-Cas9 functionality.
• It offers a scalable solution for generating diverse sgRNA libraries for advanced gene editing research.