CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid, termed prespacers, and inserting them into their CRISPR array using the proteins Cas1 and Cas2. Immediately preceding the CRISPR array is the CRISPR leader region, and prespacers are generally inserted where the leader region meets the CRISPR array (leader-repeat junction). Here, a detailed analysis of the bioinformatic, biochemical, and biophysical characteristics of the DNA and protein elements that govern this site-specific insertion of prespacers is presented. Various sequences of leader-repeat junctions were first analyzed belonging to type II-A, a sub-type of CRISPR systems. Phylogenetic clustering of leader-repeat junctions defined three distinct groups with conserved sequences, G1 with ATTTGAG, G2 with CTRCGAG, and G3 with NNNNNCG. The sequence alignment data showed phylogenetic clustering of Cas proteins and repeat sequences in type II-A systems that mirrored the clustering of leader-repeat junctions. Biochemical characterization of representative Cas1 and Cas2 proteins from each group showed distinct mechanisms in leader-repeat junction recognition and in prespacer insertion. G1 first recognized a 12-bp sequence at the leader–repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. G3 showed no sequence specific insertion activity. Protein-DNA complex formation analysis by direct molar mass measurement showed that all three protein complexes form the canonical Cas14-Cas22-prespacer1 complex, with the morphology of the prespacer being an essential factor promoting complex formation, at least in the case of G1. These results highlight the intricacy of protein–DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These insights provide valuable information for the development of a Cas1–Cas2 toolset for inserting small DNAs into diverse DNA targets.