Engineering a Cas9-monomeric Streptavidin Fusion to Increase CRISPR Knock-in Efficiency In Vitro

• Autor(a) principal: Gonçalves, Maria Filipe
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/135867
• topic: CRISPR-Cas9; Cas9-MSA; biotinylated DNA template; HDR; DR-GFP; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), a bacterial adaptative defence system, was one of the major recent biotechnological breakthroughs, which has revolutionized gene editing in several fields. This system can be artificially manipulated to guide a Cas9 endonuclease with a single guide RNA (sgRNA) to regions of interest in the genome of a given cell. When a target sequence is recognized, Cas9 inserts a double-strand break that triggers the DNA repair of that region by either the error-prone non-homologous end joining (NHEJ) or the error-free, template-dependent, homology-directed repair (HDR) pathway. To increase the local concentration of the template DNA, skewing the repair to the HDR pathway, a Cas9 fused to a monomeric streptavidin (MSA) has been used, which was tethered to a biotinylated template DNA, thus increasing the efficiency of repair. In this thesis, two-point mutations - S14R and T39W, hypothesized to increase streptavidin's affinity to biotin - were inserted into the MSA gene of the Cas9-MSA-encoding plasmid. The engineered Cas9-MSA** plasmid, as well as the original Cas9-MSA and Cas9-wild-type (WT) plasmids, were used to transfect DR-GFP cells and were shown to have comparable HDR activities. Furthermore, with the addition of an exogenous template to the transfection conditions, the mutated Cas9-MSA produced the highest number of HDR-caused events with the biotinylated template, with a 1.3-fold increase of effi-ciency when compared to the original Cas9-MSA, and a 2.1-fold increase when compared to the tradi-tional Cas9-WT system. These results are encouraging to explore this field of research further and im-prove the CRISPR-Cas9 technique to reach the required efficacy and safety for gene therapy applica-tions.