CRISPR-Cas is the state-of-the-art biological tool that allows precise and fast manipulation of the genetic information of cellular genomes. The translation of the CRISPR-Cas technology from in vitro studies into clinical applications highlighted a variety of limitations: the currently available systems are limited by their off-target activity, the availability of a Cas-specific PAM sequence next to the target and the size of the Cas protein. In particular, despite high levels of activity, the size of the CRISPR-SpCas9 editing machinery is not compatible with an all-in-one AAV delivery system and the genomic sequences that can be targeted are limited by the 3-NGG PAM-dependency of the SpCas9 protein. To further expand the CRISPR tools repertoire we turned to metagenomic data of the human microbiome to search for uncharacterized CRISPR-Cas9 systems and we identified a set of novel small Cas9 orthologs derived from the analysis of reconstructed bacterial metagenomes. In this thesis study, ten candidates were chosen according to their size (less than 1100aa). The PAM preference of all the ten orthologs was established exploiting a bacterial-based and an in vitro platform. We demonstrated that three of them are active nucleases in human cells and two out of the three showed robust editing levels at endogenous loci, outperforming SpCas9 at particular targets. We expect these new variants to be very useful in expanding the available genome editing tools both in vitro and in vivo. Knock-out-based Cas9 applications are very efficient but many times a precise control of the repair outcome through HDR-mediated gene targeting is required. To address this issue, we also developed an MS2-based reporter platform to measure the frequency of HDR events and evaluate novel HDR-modulating factors. The platform was validated and could allow the screening of libraries of proteins to assess their influence on the HDR pathway.