The CRISPR technology, specifically the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—Cas9 endonuclease system, has revolutionized genome editing in various mammalian species, including humans. This efficient method enables precise modifications in selected genes by facilitating deletions, insertions, or targeted DNA sequence modifications in a single step. Cas9, in collaboration with guide RNA, induces double-stranded breaks in the complementary DNA target, and subsequent repair processes result in the desired gene modifications. Widely adopted for genome editing and biotechnology applications, CRISPR/Cas9 serves purposes such as functional annotation, analysis of genomic loci, and regulation of gene transcription. The technique was found to be extensively used in manipulating laboratory small/large animal as well as human genomes, making substantial contributions to comprehending gene functionalities and also serving as a prominent method for modeling animal and human diseases and their therapeutics. In this regard, our chapter provides an overview of CRISPR/Cas9 technology, historical aspects, and mechanism and highlights recent advances in its application toward translational research and biomedicine.