Combinatorial strategies using CRISPR/Cas9 for gene mutagenesis in adult mice

Abstract

A major challenge to understanding how genes modulate complex behaviors is the inability to restrict genetic manipulations to defined cell populations or circuits. To circumvent this, we created a simple strategy for limiting gene knockout to specific cell populations using a viral-mediated, conditional CRISPR/SaCas9 system in combination with intersectional genetic strategies. A small single guide RNA (sgRNA) directs Staphylococcus aureus CRISPR-associated protein (SaCas9) to unique sites on DNA in a Cre-dependent manner resulting in double strand breaks and gene mutagenesis in vivo. To validate this technique we targeted nine different genes of diverse function in distinct cell types in mice and performed an array of analyses to confirm gene mutagenesis and subsequent protein loss, including IHC, cell-type specific DNA sequencing, electrophysiology, Western blots, and behavior. We show that these vectors are as efficient as conventional conditional gene knockout and provide a viable alternative to complex genetic crosses. This strategy provides additional benefits of targeting gene mutagenesis to cell types previously difficult to isolate, and the ability to target genes in specific neural projections for gene inactivation. Our studies illuminate AAV1-CMV-FLEX-SaCas9-U6-sgRNA as a valuable tool for rapid, efficient and robust analysis of gene function.