**3.1. CRISPR/CAS9 and knocking out genes in mouse**

An option to improve knockout effectiveness could be to use two or more RNA guides at the same time to knock out the oncogene allele at different key sites in an attempt to try to guarantee the null result. This approach is commonly used for knocking out genes in animal models such as mice. Using two sgRNA guides makes it possible to distinguish the mutant pups by a simple PCR.-An example of this is the generation and genetic characterization of Six6os1-deficient mice [34] (**Figure 10**).-

Unfortunately, the possibility of using several RNA guides at the same time is quite limited- in gene therapy, especially when adeno-associated virus vectors are used. The main difficulty- stems from the limitations on the construct, for which reason other Cas9 orthologues are being used to introduce the nuclease coding sequence, one promoter and a single RNA guide [26, 35].
