Barcoded Rational AAV Vector Evolution enables systematic in vivo mapping of peptide binding motifs

Engineering of Adeno-associated viral (AAV) vector capsids through directed evolution has been used to generate novel capsids with altered tropism and function1-9. This approach, however, involves a selection process that requires multiple generations of screenings to identify real functional capsids2-4. Due to the random nature of this process, it is also inherently unreproducible, and the resulting capsid variants provide little mechanistic insights into the molecular targets engaged. To overcome this, we have developed a novel method for rational capsid evolution named Barcoded Rational AAV Vector Evolution (BRAVE). The key to this method is a novel viral production approach where each virus particle displays a protein-derived peptide on the surface which is linked to a unique barcode in the packaged genome10. Through hidden Markov model-based clustering11, we were able to identify novel consensus motifs for cell-type specific retrograde transport in neurons in vivo in the brain. The BRAVE approach enables the selection of novel capsid structures using only a single-generation screening. Furthermore, it can be used to map, with high resolution, the putative binding sequences of large protein libraries.