Konstruksi Backbone Plasmid Single Target dan tRNA-gRNA (TGR) Sistem CRISPR-Cas9 untuk Tanaman

Nicholas, Farrell Wijaya (2022) Konstruksi Backbone Plasmid Single Target dan tRNA-gRNA (TGR) Sistem CRISPR-Cas9 untuk Tanaman. Diploma thesis, Universitas Andalas.

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Abstract

Plasmid construction is one of many important steps within genome editing study using CRISPR-Cas9. Variety of genes that were targeted across different organisms and research’s purposes make it necessary to acquire the backbone plasmid with multiplexing capability (tRNA-gRNA) and flexibility to change its target by modifying crRNA sequences. Moreover, the size of plasmid also affects transformation efficiency. Therefore, it becomes clear that backbone plasmid is needed to be constructed with such characteristics. Golden Gate Assembly method was chosen with a combination of PCR reaction (henceforth called Golden Gate – PCR method) to complete two overlapping objectives, mutagenesis of BsaI recognition sequences at pVS1 Ori and combining 8 separate fragments, both backbone and insert PCR products. The results show that the final plasmid has been successfully constructed with a final size of 14,728 bp. PCR verification, short to full-length plasmid, of fragments’ order showed that all parts are assembled in intended positions. Furthermore, the most efficient method for applying Golden Gate – PCR is by directly amplifying Golden Gate reaction mixture with flanking primers (Direct PCR from Golden Gate). While the PCR reaction following purification of the correct Golden Gate product (Golden Gate – Purification – PCR) also yields the desired products, it consumes two purification reactions for each assembly. Capability to assemble only small fragments is the only limitation for Golden Gate – PCR method.

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