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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.

Linsha Ma1, Pengcheng Yao2,3, Shengqi Wu1

  • 1Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops, Key Laboratory for Enhancing Resource Use Efficiency of Crops in South China, State Key Laboratory For Conservation and Utilization of Subtropical Agro-Bioresources, Guangdong Provincial Key Laboratory of Plant Molecular Breeding, Ministry of Agriculture and Rural Affairs, College of Agriculture, South China Agricultural University, Guangzhou, China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|June 26, 2026
PubMed
Summary
This summary is machine-generated.

Researchers discovered new reverse transcriptases (RTs) for CRISPR prime editors (PEs). An optimized RT variant, enRERV-RT, significantly boosts prime editing efficiency in cells and difficult genomic regions.

Keywords:
RERV (Rattus norvegicus endogenous retrovirus)TRAP‐seq‐PEcrop breedinggene therapyhard‐to‐editprime editorsprotein engineeringreverse transcriptase

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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.