2. Academic Validation
  3. Efficient targeted insertion of large DNA fragments without DNA donors

Efficient targeted insertion of large DNA fragments without DNA donors

  • Nat Methods. 2022 Mar;19(3):331-340. doi: 10.1038/s41592-022-01399-1.
Jinlin Wang  # 1 2,  Zhou He  # 1 2,  Guoquan Wang  # 1 2,  Ruiwen Zhang  # 1 2,  Junyi Duan 1 2,  Pan Gao 1 2,  Xinlin Lei 1 2,  Houyuan Qiu 1 3,  Chuanping Zhang 1 3,  Ying Zhang 1 3,  Hao Yin 4 5 6 7 8 9
Affiliations

Affiliations

  • 1 Department of Urology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China.
  • 2 Department of Pulmonary and Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China.
  • 3 Department of Rheumatology and Immunology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China.
  • 4 Department of Urology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China. [email protected].
  • 5 Department of Pulmonary and Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China. [email protected].
  • 6 Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan, China. [email protected].
  • 7 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Wuhan University School of Pharmaceutical Sciences, Wuhan, China. [email protected].
  • 8 RNA Institute, Wuhan University, Wuhan, China. [email protected].
  • 9 Wuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan, China. [email protected].
  • # Contributed equally.
PMID: 35228726 DOI: 10.1038/s41592-022-01399-1
Abstract

Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~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 ~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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