Since the isolation of adenovirus (AdV) in 1953, AdVs have been used as vectors for various therapeutic purposes, such as gene therapy in cancers and other malignancies, vaccine development and delivery of CRISPR-Cas9 machinery. Over the years, several AdV vector modifications have been introduced, including fiber switching, incorporation of ligands in the viral capsid and hexon modification of the fiber, to improve the efficiency of AdV as a vector. CRISPR-Cas9 has recently been used for these modifications and is also used in other adeno-associated viruses. These modifications further allow the production of AdV libraries that display random peptides for the production of cancer-targeting AdV vectors. This review focuses on the common methods of AdV construction, changes in AdV tropism for the improvement of therapeutic efficiency and the role of AdV vectors in gene therapy, vaccine development and CRISPR-Cas9 delivery.