Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9. Issue 1 (22nd July 2022)

• Record Type: Journal Article
• Authors: Movahedi, Ali; Wei, Hui; Zhou, Xiaohong; Fountain, Jake C; Chen, Zhong-Hua; Mu, Zhiying; Sun, Weibo; Zhang, Jiaxin; Li, Dawei; Guo, Baozhu; Varshney, Rajeev K; Yang, Liming; Zhuge, Qiang
• Is Part Of: Horticulture research. Volume 9:Issue 1(2022)
• Publication Date: 2022-07-22
• Subjects: Horticulture -- Research -- Periodicals; 635.072
• DOI: 10.1093/hr/uhac154
• Languages: English
• ISSNs: 2052-7276

Abstract

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 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 fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency showed up to 48% 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 increased genome targeting efficiency plus 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.