Abstract: To obtain mutants with large fragment deletions in the CsLOB1 promoter，in this study，we edited multiple sites of the CsLOB1 promoter using the CRISPR/Cas9 system. Two plant expression vectors，pCas9CsLOB1:2sites and pCas9CsLOB1:3sites，which were designed for the simultaneous targeting of two and three sites in the CsLOB1 promoter，respectively，were constructed to edit the EBEPthA4 region. Sequencing data revealed that the editing efficiency in pCas9CsLOB1:2sites and pCas9CsLOB1:3sites transgenic lines was 64.7% and 80.0%，respectively，and fragment deletions between two sgRNA target sites occurred in the transgenic citrus plants. Further analyses indicated the mutation efficiency differed among the sgRNAs. The differences were likely due to variability in the binding ability of sgRNAs to the targeted CsLOB1- sequence. These results showed that mutants with the deletion of large DNA fragment can be obtained through editing multiple sites within the CsLOB1 promoter.

Key words: citrus, CRISPR/Cas9 system, CsLOB1, multiple sites, gene editing