Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9
Ali Movahedi1,† , Hui Wei1,† , Xiaohong Zhou2,† , Jake C. Fountain3,† , Zhong-Hua Chen4 , Zhiying Mu2 , Weibo Sun1 , Jiaxin Zhang5 , Dawei Li1 , Baozhu Guo6 , Rajeev K. Varshney7,8, and Liming Yang1, , Qiang Zhuge,1,
1College of Biology and the Environment, Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China 2College of Forestry and Biotechnology, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China 3Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA 4School of Science, Hawkesbury Institute for the Environment, Western Sydney University, Penrith, NSW 2751, Australia 5School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210046, China 6USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA 31793, USA 7Center of Excellence in Genomics & Systems Biology, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad 502324, India 8State Agricultural Biotechnology Center, Center for Crop and Food Innovation, Food Futures Institute, Murdoch University, Murdoch, Western Australia 6150, Australia *Corresponding author. E-mail: r.k.varshney@cgiar.org,yangliming@njfu.edu.cn,qzhuge@njfu.edu.cn †Ali Movahedi,Hui Wei,Xiaohong Zhou and Jake C. Fountain contributed equally to the study.
Revised: 22 Jul 2022 Accepted: 27 Jun 2022 Published online: 06 Jan 2022
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.
and Liming Yang1 ,
