Prime editing using CRISPR-Cas12a and circular RNAs in human cells

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• CRISPR-Cas9 prime editors face limitations due to large system size and specific PAM sequence requirements.
• Existing prime editing systems are restricted in targeting and multiplexing capabilities.

Purpose of the Study:

• To develop novel prime editor systems utilizing the smaller Cas12a protein for enhanced genome editing.
• To overcome the size and PAM limitations associated with Cas9-based prime editors.
• To explore the targeting preferences and multiplexing potential of Cas12a-based prime editors.

Main Methods:

• Development of four circular RNA-mediated prime editor (CPE) systems: niCPE, nuCPE, sniCPE, and snuCPE, based on Cas12a.
• Evaluation of editing efficiencies in human cells without positive selection.
• Creation of a one-sniCPE system combining all editing components under a single promoter.
• Demonstration of multiplex gene editing by arraying multiple guide RNAs on a single circular RNA.

Main Results:

• CPE systems exhibit a preference for T-rich genomic regions and offer potential for multiplexing.
• Nuclease-based CPE systems achieved editing efficiencies up to 10.42%.
• Nickase-based CPE systems (niCPE and sniCPE) demonstrated high editing frequencies of 24.89% and 40.75%, respectively.
• Simultaneous editing of up to four genes was achieved using nickase prime editors.

Conclusions:

• Cas12a-based CPE systems provide a more versatile and efficient alternative to Cas9-based prime editors.
• The developed CPE systems overcome size and PAM limitations, enabling targeting of T-rich regions.
• The potential for multiplexing and the development of a single-component system (one-sniCPE) significantly advance genome editing technologies.