Journal article
Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9
Horticulture Research, Vol.9, Art. uhac154
2022
Abstract
CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3′ end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.
Details
- Title
- Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9
- Authors/Creators
- A. Movahedi (Author/Creator) - Nanjing Forestry UniversityH. Wei (Author/Creator) - Nanjing Forestry UniversityX. Zhou (Author/Creator) - Fujian Agriculture and Forestry UniversityJ.C. Fountain (Author/Creator) - Mississippi State UniversityZ-H Chen (Author/Creator) - Western Sydney UniversityZ. Mu (Author/Creator) - Fujian Agriculture and Forestry UniversityW. Sun (Author/Creator) - Nanjing Forestry UniversityJ. Zhang (Author/Creator) - Nanjing Normal UniversityD. Li (Author/Creator) - Nanjing Forestry UniversityB. Guo (Author/Creator) - Agricultural Research ServiceR.K. Varshney (Author/Creator) - International Crops Research Institute for the Semi-Arid TropicsL. Yang (Author/Creator) - Nanjing Forestry UniversityQ. Zhuge (Author/Creator) - Nanjing Forestry University
- Publication Details
- Horticulture Research, Vol.9, Art. uhac154
- Publisher
- Oxford University Press on behalf of Nanjing Agricultural University.
- Identifiers
- 991005543024807891
- Copyright
- © 2022 The Authors.
- Murdoch Affiliation
- Centre for Crop and Food Innovation; State Agricultural Biotechnology Centre
- Language
- English
- Resource Type
- Journal article
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- Collaboration types
- Domestic collaboration
- International collaboration
- Citation topics
- 1 Clinical & Life Sciences
- 1.54 Molecular & Cell Biology - Genetics
- 1.54.1401 Genome Editing
- Web Of Science research areas
- Genetics & Heredity
- Horticulture
- Plant Sciences
- ESI research areas
- Agricultural Sciences