Abstract
Abstract
Potatoes are beneficial for ensuring global food security and CRISPR-Cas9 gene editing technology can be used to improve numerous potato traits. This technology employs a single guide RNA (sgRNA) to direct the genome editing process mediated by the Cas9 endonuclease. Precise genome editing is achievable through careful planning and evaluation of sgRNA expression to increase the efficiency of CRISPR-Cas9 gene editing.
This project aimed to design and construct different pChimera cloning vectors harboring StU6 promoter upstream of a sgRNA scaffold using In-Fusion cloning.
Firstly, the StU6 promoter was amplified in potato Désirée and Asterix cultivars and cloned into an empty vector and E. coli cells were transformed. Then, the plasmid was isolated from the putative positive colonies, sequenced and distinct allelic variations corresponding to four promoter sequences were found in potato Désirée and Asterix cultivars.
The phylogenetic analysis of the StU6 promoters distinguished a high degree of sequence identity in Désirée and Asterix clones, and seven clones from Désirée were chosen for In-Fusion cloning. DNA fragments of seven StU6 promoters and pChimera were PCR amplified to retrieve insert and vector fragments, respectively. In-Fusion cloning was performed to clone the insert fragment into a linearized vector to create the final cloning vector containing endogenous potato StU6 promoter, and Stellar Competent Cells were transformed. Colony PCR was performed to verify putative positive colonies, but no amplification was observed.
Overall, suboptimal PCR conditions or the use of a too high insert-to-vector ratio for the In-Fusion cloning reaction may potentially be the contributing factors to the lack of amplification.
Keywords: In-Fusion cloning, CRISPR-Cas 9 gene editing, StU6 promoter, sgRNA expression, PCR, RT-qPCR, colony PCR, pChimera, potato, protoplast, Désirée, Asterix, sanger sequencing, phylogenetic analysis.
