CRISPR-mediated genome editing in poplar issued by efficient transformation

Ali Movahedi; Hui Wei; Saeid Kadkhodaei; Weibo Sun; Qiang Zhuge; Liming Yang; Chen Xu

doi:10.3389/fpls.2023.1159615

CRISPR-mediated genome editing in poplar issued by efficient transformation

Front Plant Sci. 2023 Apr 17:14:1159615. doi: 10.3389/fpls.2023.1159615. eCollection 2023.

Authors

Ali Movahedi^{1

2}, Hui Wei³, Saeid Kadkhodaei⁴, Weibo Sun¹, Qiang Zhuge¹, Liming Yang¹, Chen Xu⁵

Affiliations

¹ College of Biology and the Environment, Nanjing Forestry University, Nanjing, China.
² College of Arts and Sciences, Arlington International University, Wilmington, DE, United States.
³ Key Laboratory of Landscape Plant Genetics and Breeding, School of Life Sciences, Nantong University, Nantong, China.
⁴ Agricultural Biotechnology Research Institute of Iran, Isfahan Branch, Agricultural Research, Education and Extension Organization, Isfahan, Iran.
⁵ Nanjing Key Laboratory of Quality and Safety of Agricultural Product, Nanjing Xiaozhuang University, Nanjing, China.

Abstract

Background: CRISPR has been increasingly used for plant genetic improvements because of its high efficiency and precision. Recently, the authors have reported the possibility of homology-directed repair (HDR) using CRISPR/Cas9 through woody plants such as poplar. HDR often replaces nucleotides with one donor DNA template (DDT), including homologous sequences.

Methods: CRISPR-Cas9 was recruited, and three variables, Agrobacteria inoculator concentration, pDDT/pgRNA ratio, and homologous arm length, were designed to integrate nptII and 2XCamV 35S into the MKK2 promoter zone.

Results: Here, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by the precise integration of 2XcamV 35S and nptII, improving biochemical and phenotypic properties. Our findings confirmed that Agrobacterium inoculator OD₆₀₀ = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased MKK2 expression.

Conclusion: Efficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.

Keywords: CRISPR; DDT; efficient HDR; efficient transformation; poplar.

Grants and funding

This research was funded by Nanjing Key Laboratory of Quality and Safety of Agricultural Products (NJGS2021-16), Technological Innovation Team of Colleges and Universities of Jiangsu Province (SUJIAOKE 2021), and Key Subject of Ecology of Jiangsu Province (SUJIAOYANHAN 2022).