CRISPR/Cas9 editing of the polygalacturonase FaPG1 gene improves strawberry fruit firmness

Abstract

Firmness is one of the most important fruit quality traits in strawberries. The postharvest shelf life of this soft fruit is highly limited by the loss of firmness, where cell wall disassembly plays an important role. Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening. In this study, FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Ten independent lines, cv. “Chandler”, were obtained, and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay. The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing. The percentage of edited sequences varied from 47% up to almost 100%, being higher than 95% for seven of the selected lines. Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control, ranging from 33 to 70% increase in firmness. There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness. Minor changes were observed in other fruit quality traits, such as colour, soluble solids, titratable acidity or anthocyanin content. Edited fruits showed a reduced softening rate during postharvest, displayed a reduced transpirational water loss, and were less damaged by Botrytis cinerea inoculation. The analysis of four potential off-target sites revealed no mutation events. In conclusion, editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.

Acknowledgements

This work was supported by the Ministerio de Ciencia, Innovación y Universidades and FEDER EU funds (grant numbers AGL2017-86531-C2-1R and PID2020-118468RB-C21), and the University of Malaga (grant number B1-2020_09). CS-R was awarded a PhD Fellowship from the Ministerio de Ciencia, Innovación y Universidades (PRE2018-085509), PhD Program Advanced Biotechnology, University of Málaga. We thank Botanisches Institut (Germany) for sharing CRISPR/Cas plasmids for gene editing and Dr. Vela-Corcia (IHSM “La Mayora”-UMA-CSIC, Spain) for providing Botrytis strain B05.10.

Authors’ contributions

AJM, JM-B and JAM conceived the original project; GL-C, CS-R, CP and PDR-V performed the experiments; SP and RB-P supervised the experiments; GL-C and JAM wrote the article with the contributions of all the authors.

Data availability

The datasets generated for this study are available on request to the corresponding author.

Conflict of interests statement

The authors declare that there are no conflict of interest.

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