Clustered regulatory interspaced short palindromic repeats or CRISPR/Cas9 has emerged as a potent and versatile tool for efficient genome editing. This technology has been exploited for several applications including disease modelling, cell therapy, diagnosis, and treatment of many diseases including cancer. The in vivo application of CRISPR/Cas9 is hindered by poor stability, pharmacokinetic profile, and the limited ability of the CRISPR payloads to cross biological barriers. Although viral vectors have been implemented as delivery tools for efficient in vivo gene editing, their application is associated with high immunogenicity and toxicity, limiting their clinical translation. Hence, there is a need to explore new delivery methods that can guarantee safe and efficient delivery of the CRISPR/Cas9 components to target cells. In this review, we first provide a brief history and principles of nuclease-mediated gene editing, we then focus on the different CRISPR/Cas9 formats outlining their potentials and limitations. Finally, we discuss the alternative non-viral delivery strategies currently adopted for in vivo CRISPR/Cas9 gene editing.