The formation of flowers in higher plants is controlled by complex gene regulatory networks. The study of floral development in Arabidopsis is promoted and maintained by transposon-tagged mutant lines. In this study, we report a CRISPR/Cas9 genome-editing system based on RNA endoribonuclease Csy4 processing to induce high-efficiency and inheritable targeted deletion of transcription factors involved in floral development in Arabidopsis. Using AP1, SVP, and TFL1 as the target genes, multisite and multiple-gene mutations were achieved with a tandemly arrayed Csy4-sgRNA architecture to express multiplexed sgRNAs from a single transcript driven by the Pol II promoter in transgenic lines. Targeted deletions of chromosomal fragments between the first exon and second exon in either one or three genes were generated by using a single binary vector. Interestingly, the efficiency of site-targeted deletion was comparable to that of indel mutation with the multiplexed sgRNAs. DNA sequencing analysis of RT-PCR products showed that targeted deletions of AP1 and TFL1 could lead to frameshift mutations and introduce premature stop codons to disrupt the open-reading frames of the target genes. In addition, no RT-PCR amplified product was acquired after SVP-targeted deletion. Furthermore, the targeted deletions resulted in abnormal floral development in the mutant lines compared to that of wild-type plants. AP1 and SVP mutations increased plant branching significantly, while TFL1 mutant plants displayed a change from indeterminate to determinate inflorescences. Thus, our results demonstrate that CRISPR/Cas9 with the RNA endoribonuclease Csy4 processing system is an efficient tool to study floral development and improve floral traits rapidly and simply.

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