Fig. 3
From: Advances in designing Adeno-associated viral vectors for development of anti-HBV gene therapeutics
Directed evolution strategies of AAV capsid modification. DNA shuffling of various serotypes’ cap sequences is achieved by fragmenting the capsid sequences and assembly to create hybrid capsid sequences, which can be cloned in the a Rep-encoding plasmid to create an AAV capsid plasmid library. The plasmid is then used to package a reporter-expressing AAV genome followed by selection in vitro or in vivo. Capsid sequence random mutagenesis can be performed to create a mutant capsid library, which is cloned in to a Rep-encoding plasmid to create an AAV library that can be selected. Ancestral capsid sequences, predicted using bioinformatic tools, can be synthesized in vitro and used to produce an ancestral capsid plasmid library and an AAV library before selection