SpyCas9 ancestors’ optimisation for CRISPR-Cas toolbox refinement using in vivo models
Emilia Vivaldi
SpyCas9 ancestors’ optimisation for CRISPR-Cas toolbox refinement using in vivo models.
Rel. Clara Mattu, Miguel Angel Moreno Mateos. Politecnico di Torino, Corso di laurea magistrale in Ingegneria Biomedica, 2025
Abstract
CRISPR-Cas systems represent a breakthrough technology able to facilitate genome editing in a simple and versatile manner. Among them, class II CRISPR-Cas systems are the simplest approaches based on a protein (Cas) and a guide RNA (gRNA). First, Cas protein recognizes a sequence in the genome, called protospacer adjacent motif (PAM), and cleavages only when the sgRNA sequence specifically binds to DNA target. The most used and efficient system is CRISPR-SpyCas9 from Streptococcus Pyogenes which PAM is 5’NGG. Despite the potential of CRISPR-Cas systems, some constraints can mitigate the use of this technology. For example, PAM restriction, which does not allow free access to all sequences in the genome, together with immune response activation caused by Cas proteins in eukaryotic cells, can limit the efficiency and safeness of this technology for potential biomedical application.
To circumvent these barriers, different approaches have been developed such as the discovery of new natural CRISPR-Cas systems, the modification of known Cas or, more recently, the implementation of synthetic ancestral Cas (anCas)
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