Gene editing in the adult rat brain, using CRISPR/Cas9 to interrogate gene function, has been hampered by the size of the CRISPR-associated endonuclease SpCas9 and the low in vivo efficiency of other Cas9 homologs. Here, we present a highly efficient gene editing approach, using Cas9 from Staphylococcus aureus (SaCas9) to target the Slc18a2 gene, encoding the vesicular monoamine transporter 2 (VMAT2), in vitro and in vivo. We use in vivo positron emission tomography (PET) imaging to investigate molecular changes at pre- and postsynaptic terminals and behavioural tests to explore the phenotype.
We first selected the most efficient sgRNA targeting the Slc18a2 in vitro in rat primary neurons, using adeno-associated viral (AAV) vectors, and validated the knockdown (KD) efficiency by surveyor assay and immunofluorescence. Next, AAV-SaCas9 and AAV-sgRNA targeting Slc18a2 (n=14) or LacZ (control, n=10) were stereotactically injected into the right substantia nigra of adult rats. Starting 8 weeks after AAVs delivery, VMAT2 expression, DAT availability, postsynaptic changes and inflammatory responses, were quantified using dynamic PET imaging with [11C]DTBZ, [11C]methylphenidate (MP), [11C]raclopride (RAC) and the TSPO ligand [18F]GE-180, respectively. CRISPR-induced VMAT2 KD was further characterized with behavioral readouts of locomotor activity, gait, spontaneous and evoked rotations.
We observed a -60/+2% [11C]DTBZ decrease indicative for VMAT2 KD in the right striatum compared to the left striatum (p=0.0001). The KD did not affect [11C]MP or [18F]GE-180 binding indicating that VMAT2 expression was selectively reduced without inducing neuronal loss or inflammation. As the reduction of dopamine (DA) at presynaptic terminals is known to result in postsynaptic changes of DA receptors, we performed [11C]RAC scans and observed a 3/40% increase in the right striatum compared to the left striatum (p=0.0003). Remarkably, changes in [11C]DTBZ binding strongly correlated to changes in [11C]RAC binding (R2=0.77). We did not observe changes for the tested radioligands in the LacZ-injected group. Additionally, VMAT2 KD rats revealed significant motor alterations, partly correlating with [11C]DTBZ binding changes, in terms of paw use preference (p=0.002, R2=0.72), total distance travelled (p=0.03), gait (p=0.01), spontaneous rotation (p=0.03, R2=0.20), evoked rotation (p=0.03).
Our work pioneers the combinatorial use of CRISPR/Cas9 gene editing and PET imaging to inspect in vivo the direct link between specific genes and molecular changes observed in pathology. We used CRISPR/Cas9 to edit the Slc18a2 in the adult rat brain, reproducing motor symptoms of Parkinson´s disease. We observed an effective DA depletion in the absence of neuronal loss, which strongly correlates with DA receptors occupancy or expression changes.
Keywords: PET, CRISPR/Cas9, Dopamine, Dopamine receptors expression changes, Parkinson`s disease