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Plant Biotechnol J. 2018 Aug;16(8):1424-1433. doi: 10.1111/pbi.12884. Epub 2018 Feb 6.

Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit.

Wang Z1,2,3, Wang S1,2,3, Li D4, Zhang Q4, Li L4, Zhong C4, Liu Y1,2, Huang H1,2,4.

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Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou, Guangdong, China.
Guangdong Provincial Key Laboratory of Applied Botany, Guangzhou, Guangdong, China.
University of Chinese Academy of Sciences, Beijing, China.
Key Laboratory of Plant Germplasm Enhancement and Specially Agriculture, Wuhan Botanical Garden, The Chinese Academy of Sciences, Wuhan, Hubei, China.


Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired-sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired-sgRNA cloning, our strategy only requires the synthesis of two gRNA-containing primers which largely reduces the cost. We further compared efficiencies of paired-sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA-sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10-fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired-sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418-resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.


CRISPR/Cas9; PTG/Cas9; chromosomal fragment deletion; genome editing; kiwifruit; paired-sgRNA

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