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

Published: 9 May 2022| Version 4 | DOI: 10.17632/mnrvyvt8bs.4
Hui Wei,
Xiaohong Zhou,
Zhong-Hua Chen,
Jake C. Fountain,
Zhiying Mu,
Weibo Sun,
Jiaxin Zhang,
Dawei Li,
Baozhu Guo,
Rajeev K. Varshney,
Liming Yang,
Qiang Zhuge


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 about 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.


Steps to reproduce

1- Sanger sequencing 2- Analyzing and alignment by Geneious prime


Gene Knockout, Polymorphic Analysis, CRISPR/Cas9, Genome Editing, Sanger Sequencing