Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors

Abstract

Significance: The widespread use of recombinase-dependent adeno-associated virus (AAV) for targeting transgene expression to specific neural populations has made it a fundamental tool for neural manipulation and circuit dissection. Despite this, the well-known but oft-ignored issue of unwanted recombinase-independent “leak” expression from common recombinase-dependent DIO and FLEX AAV designs impedes certain applications and confounds data. Herein we discover that the leak expression from these constructs is due to both low-level expression of the inverted transgene and homology-dependent recombination in bacteria during plasmid production. Using this knowledge, we create a Cre-dependent AAV design that greatly decreases leak expression. This development will improve cell-type-specific transgene targeting for a wide spectrum of tools dependent on precise cell-type-specific targeting.