Engineered CRISPR prime editors with compact, untethered reverse transcriptases.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• CRISPR prime editing (PE) enables precise DNA modifications but relies on a large, fused protein complex (PE2).
• The PE2 editor comprises a nuclease (nSpCas9) and a reverse transcriptase (MMLV-RT) fused together.
• Optimizing PE editor size and enzyme compatibility is crucial for broader applications.

Purpose of the Study:

• To investigate if separated nSpCas9 and MMLV-RT proteins can function efficiently in prime editing.
• To utilize a split-protein system for rapid engineering of novel prime editor architectures.
• To expand the diversity of reverse transcriptases compatible with prime editing.

Main Methods:

• Constructing and testing a split prime editor system using nSpCas9 and MMLV-RT components in human cells.
• Employing the split system to screen for and engineer alternative, more compact prime editor designs.
• Evaluating the efficiency of different reverse transcriptase variants within the split prime editor framework.

Main Results:

• Separated nSpCas9 and MMLV-RT proteins demonstrated comparable efficiency to the intact PE2 editor in human cells.
• The split-PE system facilitated rapid identification of more compact prime editor architectures.
• The system successfully incorporated and validated alternative reverse transcriptases for prime editing.

Conclusions:

• Disassembling the PE2 prime editor into separate functional components is feasible and efficient.
• The split-prime editor system accelerates the development of optimized and versatile gene editing tools.
• This approach broadens the enzymatic toolkit for prime editing, enhancing its applicability.