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Ourlong-term goalis to develop and demonstrate appropriate technologies supporting agricultural production research to promote rural community economic vitality through agriculture. Implementing this new plant breeding technology on tropical crops will enable the manipulation of established tropical crops to be more resilient, productive, and sustainable. Besides, it can speed-up the domestication of novel tropical crops. This proposal'soverall goalis to present a transgene-free gene-editing protocol to the tropical crop community for wide-usage. Ourcentral hypothesisis that by targeting thephytoene desaturase(PDS) gene in both sugarcane and papaya, we can determine our CRISPR-Cas protocol's efficiency as edited plants will be albino (white plants).We base our hypothesis that every time the CRISPR-Cas system is tested on a new crop or strategy is implemented on a crop,PDSis the target of choice due to this simple phenotype. Furthermore, we will develop a high-throughput molecular screening to determine the presence of edited sites. Two aims are proposed to develop a transgene-free gene-editing protocol on tropical crops:Objective 1:Determine gene editing efficiencies for CRISPR-Cas9 and CRISPR-Cpf1(Cas12a) in sugarcane and papaya.Design CRISPR RNA templates targeting thephytoene desaturase(PDS) gene in sugarcane and papayaappropriate to the relevant endonuclease (i.e., Cas9 and Cas12a).Determine design effectiveness by in vitroCRISPR-Cas cleavage assay onPDS(i.e., CRISPR-Cas genotyping).Implement and optimize in-house tissue culture protocols for embryogenic callus induction, particle bombardment, calli recovery, and plantlet regeneration for the aforementioned tropical crops.Determine the optimal concentration of the gene-editing components.Determine the number of embryogenic calli generated.Determine the number of plantlets regenerated post-bombardment and recovery.Determine the number of days it takes from embryogenic callus induction to plantlet regeneration.Determine the gene-editing ratio of regenerated plantlets.Visually measure the ratio between green (wild-type or heterozygous) versus white (homozygous or allelic).Molecularly determine the genotypic classes of regenerated plantlets.Determine the alleles generated by Sanger sequencing on regenerated plantlets that showed edited events.Objective 2:Develop a high-throughput molecular screening for putative edited plantlets.Design the Locked Nucleic Acid (LNA) probe targeting the editing site appropriate to the CRISPR-Cas module used and the reference probe.Determine the optimal quantitative Polymerase Chain Reaction (qPCR) conditions for LNA assay.Test the efficiency of LNA assay on regenerated plantlets.Randomly select a subset of regenerated plantlets and confirm the genotyping call byin vitroCRISPR-Cas genotyping.

Del Valle Echevarria, A. R.; Wang, Mi, .; He, Xi, .
Hawaii Agriculture Research Center
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