Journal
NATURE METHODS
Volume 19, Issue 3, Pages 331-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41592-022-01399-1
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Funding
- National Key R&D Program of China [2019YFA0802801, 2018YFA0801401]
- National Natural Science Foundation of China [31871345, 32071442, 31972936]
- Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University [TFJC2018004]
- Fundamental Research Funds for the Central Universities
- Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences [2020-PT320-004]
- Applied Basic Frontier Program of Wuhan City [2020020601012216]
- Hubei Health Commission Young Investigator award
- Wuhan University
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GRAND editing is a technique that enables efficient insertion of large DNA fragments without the need for DNA donors. By using a pair of prime editing guide RNAs and nonhomologous reverse transcription templates, it achieves high insertion efficiency in multiple cell lines and non-dividing cells.
Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (similar to 44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to similar to 1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.
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