Abstract
The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed in the development of new crop varieties that can withstand the agricultural challenges of the 21st century. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to the multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA before in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have high in vitro cleavage activity did not produce edits in vivo, suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure the in vivo cleavage of chosen sgRNAs.
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Acknowledgements
This study was made possible through the support provided by the following institutions: the Philippine Department of Science and Technology – Science Education Institute (DOST-SEI) for the M.Sc. thesis grants of MSSagarbarria under the Accelerated Science and Technology Human Resources Development Program (ASTHDRP) and JMCaraan under the Graduate Research and Education Assistantship for Technology (GREAT) Program; the Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development (PCAARRD) project grant to the University of the Philippines Los Baños (UPLB) led by Dr. Lourdes D. Taylo (Project Code: N91832A) for the research funds. The research was also supported in part by the Plant Transgenic Design Initiative (PTraD) in the Gene Research Center at Tsukuba Plant Innovation Research Center, University of Tsukuba, Japan. We thank Dr. Kazuo N. Watanabe, who provided the sgRNA cloning vector and all-in-one Cas9/sgRNA vector. We thank Dr. Desiree M. Hautea for her research supervision in the early stages of this work. We also thank Ms. Rowena B. Frankie for her assistance with tissue culture, DNA preparation, PCR and PAGE.
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MGSS conceptualized the study and all authors contributed to the study design. JAMC performed the in vitro experiments and construct assembly. All authors performed the T7E1 assay and HMA. Transformation and regeneration were performed by MGSS and JAMC. All authors performed material preparation, data collection and analysis. The first draft of the manuscript was written by MGSS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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All experiments were conducted in accordance with the Joint Department Circular No. 1 Series of 2016 for contained use. The Department of Science and Technology Biotechnology Committee (DOST-BC) issued the corresponding Biosafety Permit No. 2020-0324. The biosafety permit conditions were complied with throughout the conduct of every experiment and associated greenhouse and laboratory activities under the supervision of the UPLB Institutional Biosafety Committee and the DA-BPI Central Post-Entry Quarantine Station.
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Sagarbarria, M.G.S., Caraan, J.A.M. & Layos, A.J.G. Usefulness of current sgRNA design guidelines and in vitro cleavage assays for plant CRISPR/Cas genome editing: a case targeting the polyphenol oxidase gene family in eggplant (Solanum melongena L.). Transgenic Res 32, 561–573 (2023). https://doi.org/10.1007/s11248-023-00371-9
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DOI: https://doi.org/10.1007/s11248-023-00371-9