An Alternative Gene Editing Strategy Using a Single AAV Vector

We recently developed an approach for cell type-specific CRISPR/Cas9 editing and transgene expression using a single viral vector. Here, we present a protocol describing how to design and generate plasmids and adeno-associated viruses (AAVs) compatible with this single-vector gene editing approach. This protocol has four components: (1) guide RNA (gRNA) design to target specific genes of interest, (2) ligation and cloning of CRISPR-competent AAV vectors, (3) production of vector-containing AAVs, and (4) viral titer quantification. The resultant vectors are compatible for use with mouse lines expressing the Cas9 protein from <i>Streptococcus pyogenes</i> (SpCas9) and Cre recombinase to enable selective co-expression of standard neuroscience tools in edited cells. This protocol can produce AAVs of any serotype, and the resulting AAVs can be used in the central and peripheral nervous systems. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection. Key features • Single-vector CRISPR/Cas9 gene editing and genetically encoded tool delivery for use in mouse central and peripheral nervous systems. • Can be combined with many genetically encoded tools, including fluorescent proteins, optogenetic and chemogenetic tools, and calcium imaging. • Requires first-generation cross between a Cre driver mouse line and Cre-dependent SpCas9 mouse line. • Optimized for use with SpCas9.