In order to study the role of certain proteins on regeneration we need to manipulate their function. We need to reduce/increase their function and monitor its effect on regeneration. Therefore, we need to generate certain DNA constructs. We use classic restriction-ligation cloning as well as HiFi DNA assembly. The constructs that we generate are dominant negative forms and constitutive active forms of our molecules of interest. Expression of dominant negative constructs will negatively affect the wild type protein function within the same cell and reduce its activity, while expression of constitutive active forms will increase activity at the cellular level. These techniques are very useful however, they are based on overexpression and might result in overexpression artifacts. For this purpose, we are also using CRISPR/Cas9 mediated genome targeting. CRISPR/Cas9 enables us to directly manipulate the endogenous locus and as such we can generate loss-of-function and gain-of-function alleles of our gene of interest, without having to resort to overexpression assays.