Few scientific discoveries are as transcendent as the CRISPR-Cas9 genetic engineering technique. This technique enlarges its potential when combining it with ancestral reconstruction. During this work, 5 different Ancestral Cas9 enzymes have been characterized by following the Ancestral sequence reconstruction (ASR) technique which ...»»»»
Few scientific discoveries are as transcendent as the CRISPR-Cas9 genetic engineering technique. This technique enlarges its potential when combining it with ancestral reconstruction. During this work, 5 different Ancestral Cas9 enzymes have been characterized by following the Ancestral sequence reconstruction (ASR) technique which allows us going back in time.
The main purpose of this work is using these reconstructed Ancestral Cas9 enzymes to analyse their activity by performing in vitro assays. In order to achieve success during these experiments, the synthetized ancestral Cas9 are cloned in a plasmid and transferred to bacteria for their posterior growth and purification by using the Fast protein liquid chromatography (FPLC). Essential elements in CRISPR-Cas9 as the guide RNA and the target DNA library are respectively synthetized and linearized for increasing the effectiveness of the assays. Another crucial element is the Protospacer Adjacent Motif (PAM) which needs to be recognized by the enzyme to perform the desired cleavage.
These assays permits us to observe that even suffering hundreds of mutations, the enzymes maintain their catalytic activity and are able to join sgRNA and cleave DNA target after recognizing a PAM sequence. Taking into account the limitations that this technique could have, the future challenge of this work would be its total characterization and application in vivo treatments.^^^^
