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CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes

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Abstract

Main conclusion

VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes.

Abstract

CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90–99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars’ analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.

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Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

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Authors and Affiliations

  1. Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Bank Road, Lahore, 53700, Pakistan

    Aneela Yasmeen, Sana Shakoor, Saira Azam, Allah Bakhsh, Naila Shahid, Ayesha Latif, Ahmad Ali Shahid, Tayyab Husnain & Abdul Qayyum Rao

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  1. Aneela Yasmeen
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  2. Sana Shakoor
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  3. Saira Azam
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  4. Allah Bakhsh
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  5. Naila Shahid
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  6. Ayesha Latif
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  7. Ahmad Ali Shahid
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  9. Abdul Qayyum Rao
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Contributions

AY: performed all the experiments related to vector confirmation, genetic transformation, compiled data and wrote initial draft of manuscript. AQR: designed study, constructed recombinant plasmids, provided technical support, and supervised the study. SA, NS and AL: helped in genetic transformation experiments and recorded data. AB: helped in mutant screening and phenotypic analysis. SS: helped in morphological and physiological assays, AAS: edited the manuscript and interpreted data, TH: supervised the overall study, read the manuscript critically and presented it in its current form.

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Correspondence to Abdul Qayyum Rao.

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Communicated by Anastasios Melis.

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Yasmeen, A., Shakoor, S., Azam, S. et al. CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes. Planta 256, 107 (2022). https://doi.org/10.1007/s00425-022-04022-x

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