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Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9

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Relation http://oar.icrisat.org/12660/
https://academic.oup.com/hr/article/doi/10.1093/hr/uhac154/6648883
https://doi.org/10.1093/hr/uhac154
 
Title Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9
 
Creator Movahedi, A
Wei, H
Zhou, X
Fountain, J C
Chen, Z
Mu, Z
Sun, W
Zhang, J
Li, D
Guo, B
Varshney, R K
Yang, L
Zhuge, Q
 
Subject Genetics and Genomics
 
Description 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.
 
Publisher Oxford University Press on behalf of Nanjing Agricultural University
 
Date 2022-07-22
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
Rights cc_attribution
 
Identifier http://oar.icrisat.org/12660/1/Horticulture%20Research_9_1-15_2022.pdf
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