Development and application of genetic engineering tools

• Main Author: Teo, Yi Yong
• Other Authors: Tan Meng How
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Engineering::Bioengineering
• Online Access: https://hdl.handle.net/10356/159073
• Institution: Nanyang Technological University

SNPs in the genomic DNA are linked to numerous diseases. In these cases, the disease and healthy alleles differ by a single DNA base. Using CRISPR-Cas9 base editors, it is possible to modify SNPs by converting the targeted disease-related DNA base into another base. CGBEs are different from CBEs and ABEs, and can be used to convert C:G base pairs to G:C base pairs. CGBEs can be packaged into AAV vectors as a means of delivery to reach the target cells in vivo. AAV vectors are used due to its low pathogenic properties. However, AAV vectors have a packaging limit of ~5kb. This means that the CGBE to be packaged into the AAV vector needs to be below this packaging limit to have the intended function. Thus, there is a need to truncate the CGBE such that it can be packaged into the AAV vector. However, truncating the CGBE would mean that segments of DNA in CGBE would have to be removed. Therefore, to ensure that the CGBE construct would continue to function as intended after the removal of the DNA segments, the editing efficiencies of the truncated CGBE are investigated. Different deletions of DNA segments at various positions of the CGBE construct are also investigated to generate a truncated CGBE with relatively high editing efficiency. The experiment determined the combinations of deletions in relation to the editing efficiency of the contructs.