An improved plant CRISPR-Cas9 system for generating Cas9-free multiplex mutants

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated gene editing systems have been used in a variety of organisms, including plants. This system can efficiently edit target genes to generate mutants, especially multiplex mutants, which are invaluable genetic materials for gene functional studies. Although the use of traditional hybridization technology to construct multiplex mutants has been widely used in plant research, the complex screening and identification, long construction cycle and other problems have always troubled researchers. Therefore, in the current plant science research, the use of CRISPR-Cas9 mediated multiplex genome editing to construct multiplex mutants has become a mainstream technology. Here, we designed an improved plant CRISPR-Cas9 mediated multiplex genome vector system. The In-fusion cloning technology was selected to conveniently assemble multiple tandem sgRNA (Single-guide RNA) expression frames. In this system, Cas9-free plants can be easily selected by examining mCherry fluorescence in T-2 transgenic plants. By using this system, Cas9-free homozygous triple mutant was obtained, demonstrating the availability of our system.