Abstract
Perineuronal nets (PNNs) are specialized extracellular matrix structures enwrapping a subset of neurons in the central nervous system. PNNs are thought to play an important regulatory part in the mechanisms of learning and memory through their ability to stabilize synapses and restrict neuroplasticity, and the disruption of these structures has been linked to several neurological and psychiatric disorders. Still, the underlying mechanism of the function of PNNs remains largely unknown. In the pursuit to gain more knowledge of PNNs, it would be of great value to facilitate the study of PNNs over time in vivo by fluorescent tagging of PNN components. This could be achieved by performing knock-in (KI) of a fluorophore DNA sequence into the genome of neurons. One promising new genome engineering technology that offers a fast and cost efficient method of achieving this goal is CRISPR-Cas9 technology (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated protein 9). The main aims of this thesis were therefore local optimization of the CRISPR-Cas9 KI method and to apply this technology for fluorescent tagging of aggrecan, one of the main PNN components. As a starting point for the project, a published adeno-associated virus vector with CRISPR elements for KI of a red fluorophore sequence targeting the gene for β-actin (Actb) was obtained. To implement the KI method, pilot experiments testing the obtained construct were conducted before the start of this thesis, with the results displaying KI in mice with endogenous Cas9 expression. Still, there were a few limitations that prompted further experiments with the aim of optimizing the method. We tried to increase the KI efficiency by increasing the Cas9 expression and the transduction concentration of the KI virus. In addition, we sought to increase the brightness of the fluorescent KI signal by changing the original fluorophore to a brighter one. For the fluorescent tagging of aggrecan, we generated a CRISPR KI construct targeting the aggrecan-encoding gene Acan. After producing virus with the Acan KI construct, we tested the virus in wild-type mouse organotypic brain slice cultures. Although we established an infrastructure for testing and improving the CRISPR-Cas9 KI method, there were suboptimal experimental conditions and uncertainties concerning the Cas9 expression that rendered us unable to draw a decisive conclusion from the results. Based on all experiments, we propose a specific set of next steps for further work on implementation of the technology.