Abstract:

【Objective】 The CRISPR/Cas9 ribonucleoprotein (RNP) system is an efficient gene editing technology that is simple and accurate and has been widely used in animal and plant gene editing research. Introducing the Cas9 protein and gRNA RNP complex into recipient cells by particle bombardment to obtain marker-free gene-edited plants provides an effective approach for the rapid plant mutant creation. In this study, the BpGLK1 gene of birch (Betula platyphylla × B. pendula) was used as the target gene for editing, and CRISPR/Cas9 RNP technology was used to edit the BpGLK1 gene without T-DNA insertion. 【Method】 A target site was designed for the first exon of birch BpGLK1. The target fragment of BpGLK1-E1 was amplified by PCR. The recombinant plasmid pAbAi-BpGLK1-E1 was constructed by enzyme digestion and ligation, whereafter the plasmid was linearized and reacted with Cas9 protein to detect the CRISPR/Cas9 RNP activity and gRNA accuracy in vitro. Zygotic birch embryo-induced calluses were used as receptor for BpGLK1 directed mutagenesis via particle bombardment. 【Result】 The in vitro CRISPR/Cas9 RNP activity assay indicated that the Cas9 protein and gRNA could effectively cleave specific BpGLK1 target sites. The mature birch embryos were bombarded with gold particles coated with Cas9 protein and gRNA (RNP) at a distance of 12 cm and 7 584.5 kPa after 25 d of culture without light. After differentiation and subculturing, a glkc mutant with yellow-green leaves was obtained. Sequencing results showed that the mutant had a homozygous deletion of 18 bp in BpGLK1 gene. 【Conclusion】 A precise birch BpGLK1 mutation without T-DNA insertion was achieved using CRISPR/Cas9 editing technology.

Key words: CRISPR/Cas9 system, no T-DNA insertion, gene editing, Betula platyphylla×B. pendula