Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles.
Rational design of improved gene delivery vehicles is a challenging and potentially time-consuming process. As an alternative approach, directed evolution can provide a rapid and efficient means for identifying novel proteins with improved function. Here we describe a methodology for generating very large, random adeno-associated viral (AAV) libraries that can be selected for a desired function. First, the AAV2 cap gene is amplified in an error-prone PCR reaction and further diversified through a staggered extension process. The resulting PCR product is then cloned into pSub2 to generate a diverse (>10(6)) AAV2 plasmid library. Finally, the AAV2 plasmid library is used to package a diverse pool of mutant AAV2 virions, such that particles are composed of a mutant AAV genome surrounded by the capsid proteins encoded in that genome, which can be used for functional screening and evolution. This procedure can be performed in approximately 2 weeks.
Koerber
James T
JT
Department of Chemical Engineering and the Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720-1462, USA.
Maheshri
Narendra
N
Kaspar
Brian K
BK
Schaffer
David V
DV
D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D006801:Humans; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods