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

Authors: MOVAHEDI, ALI - Nanjing Forestry University; WEI, HUI - Nanjing Forestry University; ZHOU, XIAOHONG - Zhejiang A & F University; FOUNTAIN, JAKE - Mississippi State University; CHEN, ZHONG-HUA - Western Sydney University; MU, ZHIYING - Zhejiang A & F University; SUN, WEIBO - Nanjing Forestry University; ZHANG, JIAXIN - Nanjing Forestry University; LI, DAWEI - Nanjing Forestry University; Guo, Baozhu; VARSHNEY, RAJEEV - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; YANG, L - Nanjing Forestry University; QIANG, ZHUGE - Nanjing Forestry University

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2022
Publication Date: 7/22/2022
Citation: 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., Qiang, Z. 2022. Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9. Horticulture Research. 9, uhac154. https://doi.org/10.1186/s12870-023-04041-0.
DOI: https://doi.org/10.1186/s12870-023-04041-0

Interpretive Summary: CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing has become a powerful tool for 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. The objective of this study was to examine the effects of HDR cofactor overexpression and simultaneous disruption of non-homologous end joining (NHEJ) promoter on knock-in efficiency. This study demonstrated that NHEJ factor deficiency, together with HDR factor overexpression, resulted in enhanced HDR efficiency up to a 40-fold increase and dramatically expanded the capacity for trait improvement in plants. This method using HDR enhancer overexpression and NHEJ repression also resulted in 7-fold fewer CRISPR-induced Insertions and Deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses. Therefore, this approach may be useful not only in poplar and plants but also in mammalians for improving CRISPR-mediated gene knock-in efficiency.

Technical Abstract: CRISPR-mediated genome editing has become a powerful tool for 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 the 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 exogenous BleoR expression, the HDR-mediated knock-in efficiency was up to ~40-fold greater when using a XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared to no HDR enhancement or NHEJ silencing. Furthermore, this corporation of HDR enhancer overexpression and NHEJ repression also resulted in 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 mammalians for improving CRISPR-mediated gene knock-in efficiency.