Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• CRISPR-based prime editors (PEs) enable precise DNA modifications without double-strand breaks.
• The efficiency of PEs relies heavily on reverse transcriptases (RTs), with a limited number of highly active candidates available.
• Developing improved RTs is crucial for advancing PE technology.

Purpose of the Study:

• To identify novel, highly active RTs for enhancing CRISPR prime editor efficiency.
• To engineer an optimized RT variant with superior performance compared to existing systems.
• To establish a high-throughput platform for systematic evaluation of prime editor performance.

Main Methods:

• Screening of 558 RT candidates to identify novel active enzymes.
• Structure-guided engineering and deep mutational scanning to optimize RT variants.
• Development of a high-throughput platform (TRAP-seq-PE) for performance evaluation.

Main Results:

• Identified 19 novel active RTs, with RERV-RT from Rattus norvegicus showing the highest activity.
• Developed an optimized variant, enRERV-RT, which demonstrated 1.20-fold higher efficiency in mammalian and plant cells compared to M-MLV-RT based systems.
• Achieved 1.88-fold higher efficiency at hard-to-edit loci and enabled precise multiplex editing.
• TRAP-seq-PE platform confirmed higher editing efficiencies for enRERV-RT based PEs across diverse mutation types.

Conclusions:

• A versatile, high-efficiency prime editing system based on enRERV-RT has been established.
• This optimized system significantly enhances gene editing capabilities, particularly at challenging genomic sites.
• The findings facilitate advancements in clinical gene therapy and precise crop breeding through improved gene editing tools.