Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (5): 873-882.doi: 10.16420/j.issn.0513-353x.2020-1829

• Research Papers • Previous Articles     Next Articles

Establishment of Dual-cut CRISPR/Cas9 Gene Editing System in Pear Calli

YANG Feng, YANG Qinsong, GAO Yuhao, MA Yunjing, XU Ying, TENG Yuanwen, BAI Songling*()   

  1. Department of Horticulture,College of Agriculture and Biotechnology,Zhejiang University,State Agriculture Ministry Key Laboratory of Horticultural Plant Growth,Development & Quality Improvement,Hangzhou 310058,China
  • Received:2020-10-10 Revised:2021-01-26 Online:2021-05-25 Published:2021-06-07
  • Contact: BAI Songling


This work aims to establish the CRISPR/Cas9 gene editing system using European pear calli,and provides a technical platform for functional genomics study and molecular breeding of pear. We edited theGUS gene in proDAM3:GUS transgenic pear calli using a dual-cut strategy. Based on the sequence characteristics of the GUS gene,two sgRNAs were designed to target the second exon of the GUS gene. The corresponding GUS-pYLCRISPR/Cas9 plasmids were constructed and transformed into the proDAM3:GUS transgenic calli by Agrobacterium-mediated transformation. The results from sequencing data revealed that four out of six independent transgenic lines carried mutations at the target sites,with mutation rates of 66.7%. A total of 56 successfully sequenced clones from the gene edited lines were generated,which indicated that sgRNA1 target had the higher mutation frequency(71.4%)than that of sgRNA2 target(46.4%). We further analyzed the mutation types of the four GUS editing lines,and found that #1,#2,#3 lines showed deletion and insertion of nucleotides,while #5 lines showed deletion of large fragments between two target sites,whereas no nucleotide substitution was observed in any edited line. To check the phenotype,GUS staining showed that the control calli were blue in color,while the four gene editing lines were white,indicating that the CRISPR/Cas9 system was a powerful and precise method to induce targeted mutagenesis in pear calli.

Key words: pear, calli, gene editing, multiple target sites, CRISPR/Cas9

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