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bioRxiv · 10.1101/2020.07.04.188219

CRISPR-Cas9 integrates exogeneous sorting new recombinant DNA in the model tree Populus trichocarpa

Movahedi, A.·Wei, H.·Chen, Z.-H.·Sun, W.·Zhang, J.·Li, D.·Ruan, H.·Zhuge, Q.
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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.

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Movahedi, A., Wei, H., Chen, Z.-H., Sun, W., Zhang, J., Li, D., Ruan, H., Zhuge, Q.. 2020-07-06. CRISPR-Cas9 integrates exogeneous sorting new recombinant DNA in the model tree Populus trichocarpa. https://doi.org/10.1101/2020.07.04.188219

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