AAV capsids have 9 surface-exposed variable regions (VRs), named VR-I to VR-IX, that are critical for determining tropism and receptor binding. Current efforts in AAV capsid library-directed evolution primarily target VR-VIII, with some focus on VR-IV. VR-VIII, particularly the region around the spike at the 3-fold axis (e.g., N587/R588 of AAV2, or Q588/A589 of AAV9), is a common engineering site. Libraries evolved at this region have yielded well-known capsids such as AAV2-7M8, AAV-PhP.eB, AAV-Cap.Mac, and MyoAAVs.

VR-IV, the farthest protruding surface loop, plays a crucial role in neutralizing antibody binding and receptor interactions due to its proximity to the 3-fold symmetry axis. While VR-IV is less permissive to peptide insertions compared to VR-VIII, introducing diversity into VR-IV can enhance its interaction with VR-VIII mutations and refine transduction efficiency. A notable example is AAV.Cap.B22, which was engineered from AAV-PhP.eB. Directed evolution of the AAV6 VR-IV library alone led to the development of Ark313, which shows high transduction efficiency in murine T cells.

To date, few capsid engineering efforts have involved other VRs, though they do contribute to the structural and functional diversity of AAVs. VR-V, together with VR-IV and VR-VIII, forms the top of the 3-fold protrusion; VR-II forms the top of the 5-fold channel; VR-VI and VR-VII shape the base of the 3-fold protrusion; and VR-I, VR-III, VR-VII, and VR-IX contribute to the 2/5-fold wall. These VRs are essential for functional differences in receptor attachment,