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Genome-wide specificity of prime editors in plants.
Shuai Jin1,2, Qiupeng Lin1,2, Yingfeng Luo3
1State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
Nature Biotechnology
|April 16, 2021
Summary
Prime editors (PEs) offer precise genome editing but require specificity evaluation. This study found low off-target edits in plants, confirming PEs
Area of Science:
- Plant biotechnology
- Molecular biology
- Genome editing
Background:
- Prime editors (PEs) are advanced genome editing tools with therapeutic, agricultural, and research potential.
- Comprehensive evaluation of PE specificity, particularly in plants, is crucial for their safe application.
Purpose of the Study:
- To systematically assess the specificity and potential off-target effects of prime editors in plant cells.
- To evaluate the impact of mismatches in prime editing guide RNA (pegRNA) on editing efficiency.
- To determine if PEs induce genome-wide off-target mutations or alter genomic structures.
Main Methods:
- Examined mismatch tolerance of PEs in plant cells by analyzing editing frequencies based on pegRNA mismatches.
- Assessed PE activity at 179 predicted off-target sites using 12 different pegRNAs.
- Performed whole-genome sequencing on 29 PE-treated rice plants.
- Evaluated the effect of Moloney murine leukemia virus reverse transcriptase expression on retrotransposon copy number, telomere structure, and sequence insertions.
Main Results:
- Editing frequency was influenced by the number and location of mismatches in the pegRNA's primer binding site and spacer.
- Low frequencies of off-target edits (0.00–0.23%) were detected at predicted off-target sites.
- Whole-genome sequencing confirmed no induction of genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions.
- Ectopic expression of the reverse transcriptase component did not alter retrotransposon copy number, telomere structure, or lead to pegRNA/mRNA insertions.
Conclusions:
- Prime editors exhibit high specificity in plant cells with minimal detectable off-target mutations.
- The study provides crucial data supporting the safety and reliability of prime editors for plant genome editing applications.
- PEs do not appear to induce unintended genomic alterations, making them a promising tool for crop improvement and research.
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