A CRISPR/Cas9 Toolbox for the Rapid Generation of Intersectional Dual Recombinase Mouse Lines for Anatomical, Molecular, and Functional Neural Circuit Mapping

During development, many distinct neuronal and glial subtypes arise and intercalate to create the complex neural circuits that govern our behavior and physiology. Key to understanding this organization and how it may be perturbed in disease is the ability to identify and access discrete cell subtypes in the developing and adult mouse nervous systems for study. It is now clear that even within narrowly defined cell types once thought as homogeneous, significant diversity is found at multiple levels including genetic and molecular signatures, activity patterns, and synaptic connectivity and projection patterns. Recent innovations in intersectional genetics allow targeting of subtypes of neurons defined by two distinct properties (gene expression domain, spatial‐anatomical domain, temporal domain, etc.) that are transduced by dual‐recombinase Cre and FLP activities, refining the cellular population being studied. Because of the complexity of the dual recombinase conditional expression cassettes, generation of intersectional mouse lines is often unwieldy and time‐consuming, limiting the number of lines that are available. To facilitate the rapid generation of intersectional mouse lines, we developed a highly modular vector construction system that also takes advantage of CRISPR/Cas9 mediated homology directed repair (HDR) to target the Rosa26 locus with intersectional cassettes in mouse embryonic stem (ES) cells. We found that by co‐electroporating a circular vector expressing Cas9 and a Rosa26‐specific sgRNA, we could target complex and repetitive cassettes to the Rosa26 locus in ES cells using a simplified targeting vector with a 5–10 fold increase over traditional ES cell targeting (6–14% targeting vs. 58–63%). To achieve even greater efficiencies in mouse generation, we were able to multiplex five different cassettes ranging from 6.7–11kb in a single electroporation with successful targeting of all five cassettes and subsequent germline transmission. The new modular system as well as our earlier efforts have now yielded several new intersectional mouse lines and a library of ES cells for later mouse line derivation. These resources will permit functional, molecular, and anatomical characterizations of the same precise intersectionally defined neuronal populations by expressing a variety of genetically encodable effector molecules that enable functional neuronal perturbations, affinity purification of nucleic acids and proteins, and highlight specific anatomical features and network dynamics. In conclusion, our results demonstrate that CRISPR/Cas9 can be used to increase targeting efficiency at the Rosa26 locus in mouse embryonic stem cells with shortened 1.2–1.3 kb homology arms while preserving pluripotency and the ability to transmit alleles via the germline, furthering the opportunity to rapidly generate needed mouse lines for multivariate studies of neural circuit organization in physiology and behavior. Support or Funding Information NHLBI R01 HL130249, BCM McNair Scholar Program, March of Dimes Basil O'Connor Research Award, Parker B. Francis Fellowship, Dunn Collaborative Research Award, CJ Foundation for SIDS, American Heart Association