A simple and reliable method for creating PCR-detectable mutants in Arabidopsis with the polycistronic tRNA–gRNA CRISPR/Cas9 system

Abstract

Abstract To develop an easy and robust method for creating genetically stable and easily detectable Arabidopsis mutants, we adopted the polycistronic tRNA–gRNA CRISPR/Cas9 (PTG/Cas9) system, a multiplex gene-editing tool in rice, with PTOX as the reporter gene. The PTG/Cas9 system has a great potential in generating large deletions detectable by PCR, which greatly simplifies the laborious work of mutant screening. We constructed a PTOX–PTG/Cas9 system with five gRNAs and introduced it into Arabidopsis. At T1 generation, 24.4% of transgenic plants were chimeric with PCR-detectable deletions in PTOX locus, but no homozygous mutant was found, indicating that gene editing occurred predominantly in somatic cells. After a self-cross propagation, 60% of T1 chimeric plants were able to produce homozygous, heterozygous, or bi-allelic ptox offsprings. Inheritable homozygous ptox mutants without Cas9 gene can be obtained earliest at T2 generation. We further targeted five other genes using the same procedure and achieved homozygous Cas9-free mutants with large deletions for all genes within three generations. We established a standard and reliable protocol to generate stable inherited deletion mutants in 2–3 generations along with simple PCR screening methods. We conclude that the rice PTG/Cas9 system is an efficient, easy, and rapid tool to edit genes in Arabidopsis. We propose that it could be applied to other genes in Arabidopsis, and it might have the potential to edit genes in other plant species as well.