Movahedi, A and Wei, H and Zhou, X and Fountain, J C and Chen, Z and Mu, Z and Sun, W and Zhang, J and Li, D and Guo, B and Varshney, R K and Yang, L and Zhuge, Q (2022) Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9. Horticulture Research, 9. pp. 1-15. ISSN 2052-7276
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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.
| Item Type: | Article |
|---|---|
| Divisions: | Center of Excellence in Genomics and Systems Biology |
| CRP: | UNSPECIFIED |
| Uncontrolled Keywords: | CRISPR-Cas9, NHEJ suppression, HDR overexpression |
| Subjects: | Others > Genetics and Genomics |
| Depositing User: | Mr Nagaraju T |
| Date Deposited: | 07 May 2024 05:22 |
| Last Modified: | 07 May 2024 05:22 |
| URI: | http://oar.icrisat.org/id/eprint/12660 |
| Official URL: | https://academic.oup.com/hr/article/doi/10.1093/hr... |
| Projects: | UNSPECIFIED |
| Funders: | UNSPECIFIED |
| Acknowledgement: | This project was funded by the National Key Program on Transgenic Research (2018ZX08020002) and the National Natural Science Foundation of China (31971682, 31570650, and 2045210646). |
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