An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept

Abstract

A major issue in neuroscience is the poor translatability of research results from preclinical studies in animals to clinical outcomes. Comparative neuroscience can overcome this barrier by studying multiple species to differentiate between species-specific and general mechanisms of neural circuit functioning. Targeted manipulation of neural circuits often depends on genetic dissection, and use of this technique has been restricted to only a few model species, limiting its application in comparative research. However, ongoing advances in genomics make genetic dissection attainable in a growing number of species. To demonstrate the potential of comparative gene editing approaches, we developed a viral-mediated CRISPR/Cas9 strategy that is predicted to target the oxytocin receptor ( Oxtr ) gene in >80 rodent species. This strategy specifically reduced OXTR levels in all evaluated species ( n = 6) without causing gross neuronal toxicity. Thus, we show that CRISPR/Cas9-based tools can function in multiple species simultaneously. Thereby, we hope to encourage comparative gene editing and improve the translatability of neuroscientific research.

Strategies for enhanced gene delivery to the central nervous system

“…AAV vectors are widely used for delivering CRISPR/Cas9 gene-editing tools in vivo due to their ability to efficiently transduce cells and their powerful capability of delivery DNA to the cell nucleus. 27 However, limitations associated with AAVs for CRISPR/Cas9 delivery still required effort to overcome: limited cargo capacity: AAV vectors have a restricted packaging capacity, limiting the size of the genetic cargo they can carry. This constraint poses challenges when delivering large CRISPR/Cas9 components, such as long guide RNAs or Cas variants with additional functionalities.…”