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Developing a multi-modular assembled prime editing (mPE) system improved precise multi-base insertion efficiency in

Pengjun Lu1, Erwei Zuo1, Jianbin Yan1

  • 1Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China.

Journal of Advanced Research
|June 28, 2024
PubMed
Summary
This summary is machine-generated.

Prime Editing (PE) efficiency in dicots is improved by splitting the system into a multi-modular assembled prime editing system (mPE). This novel approach enhances precise editing and multi-base insertions, overcoming limitations of the original PE architecture.

Keywords:
Arabidopsis thalianaCRISPRDicotGenome editingNicotiana benthamianaPrime editing

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Area of Science:

  • Plant Synthetic Biology
  • Genome Editing Technologies
  • Molecular Biology

Background:

  • Prime Editing (PE) is a precise genome editing tool with significant potential in plant breeding and synthetic biology.
  • Low PE efficiency in dicotyledonous plants (dicots) currently limits its application.
  • Existing PE systems struggle with undetectable and inconsistent editing in dicots, hindering stable transformation.

Purpose of the Study:

  • To investigate and address the low efficiency of Prime Editing in dicots.
  • To develop and optimize a split PE system for improved performance in dicotyledonous plants.
  • To identify the optimal split PE configuration for enhanced gene editing in dicots.

Main Methods:

  • Large-scale Agrobacterium-mediated transformation experiments were conducted in Arabidopsis thaliana and Nicotiana benthamiana.
  • Deep amplicon sequencing (0.2-0.5 million clean reads) was employed to analyze editing efficiency.
  • Systematic evaluation of split PE components, including fused reverse transcriptase and extended pegRNA, was performed.

Main Results:

  • The original fused PE architecture showed reduced editing efficiency in dicots.
  • A novel multi-modular assembled prime editing system (mPE) was developed, demonstrating significantly improved editing efficiency.
  • mPE achieved editing efficiency increases ranging from 1.3-fold to 1288.5-fold, enabling editing at previously inaccessible sites and excelling in multi-base insertions (average 197.9-fold improvement).

Conclusions:

  • The original PE architecture negatively impacts Cas9 cleavage activity in dicots.
  • Split PE systems, particularly the developed mPE, substantially enhance editing efficiency in dicot plants.
  • The mPE system shows particular promise for introducing small insertions, highlighting the specialized capabilities of different PE variants.