🧫 Experiment Protocol
In VivoAlzheimer's diseaseTYROBP5xFAD x Cx3cr1-Cre mice injected ICV with AAV9-flex-dCas9-KRAB-sgTYROBP at P60abandoned
Determine whether microglia-specific, Cre-inducible CRISPRi knockdown of TYROBP (50-70%) at early amyloid onset (2 months) delays synaptic loss and cognitive decline without impairing baseline microglial homeostasis in 5xFAD mice.
PRIMARY OUTCOME
>=40% TYROBP knockdown in microglia; >=20% preservation of synaptic puncta at 6 months vs 5xFAD-vehicle
EXPECTED OUTCOMES
CRISPRi will reduce TYROBP 50-70%, shift microglia away from DAM-high inflammatory states, preserve synaptic density by >=20%, and improve NOR/Barnes maze performance.
SUCCESS CRITERIA
Primary: TYROBP knockdown >=50% confirmed by qPCR in CD11b+ cells. Secondary: PSD-95 puncta >=20% higher than 5xFAD-scramble (p<0.05).
PROTOCOL
1. Construct AAV9-flex-dCas9-KRAB with 3 sgRNAs targeting TYROBP exon 2. 2. ICV injection at P60 (2 uL, 5e12 vg/mL) in 5xFAD x Cx3cr1-Cre and WT-Cre litters. 3. Groups (n=12 each): WT-Cre-AAV, 5xFAD-Cre-scramble, 5xFAD-Cre-sgTYROBP. 4. Monthly: open field, NOR; at 6 months: CFC, Barnes maze. 5. qRT-PCR (TYROBP in CD11b+ cells), sc-RNA-seq of sorted microglia, synaptophysin/PSD-95 IHC, Abeta IHC, complement receptor C1q staining. 6. Primary: TYROBP mRNA (qPCR); secondary: PSD-95 puncta, cognition scores.
Source: task:a989715e-c687-4558-91ca-74fce1474bd2