A method for generating genome edited plant lines from CRISPR-transformed Shanxin poplar plants

Abstract

Due to the reason of low efficiency of mutation in CRISPR-editing, a high frequency of CRISPR transformed plant lines failing in mutation had been generated and had to be discarded. In the present study, we built a method to increase the efficiency of CRISPR-editing. We used Shanxin poplar (Populus davidiana×P. bolleana) as the study material, and CRISPR-editing system was first built to generate the CRISPR-transformed lines. The line that failed in CRISPR-editing was used for improving the efficiency of mutation, which was treated with heat (37 °C) to improve the cleaving activity of Cas9, leading to increased frequency of the cleaved DNA. Our results indicated that 87–100% of cells in CRISPR-transformed plants whose DNA had been cleaved by heat treatment, and the heat treatment plants were then cut into explants to differentiate adventitious buds. Each differentiated bud can be considered as an independent line. Twenty independent lines were randomly selected for analysis, and all of them had been mutated by CRISPR editing, displaying 4 types of mutation. Our results indicated that heat treatment combined with re-differentiation can generate CRISPR-edited plants efficiently. This method could conquer the problem of low mutation efficiency of CRISPR-editing in Shanxin poplar, and will have a wide application in plant CRISPR-editing.