Conditional TYROBP Knockdown in Early-Stage 5xFAD Mice Using CRISPRi-AAV9

In_Vivo Score: 0.746 Price: $0.50 Alzheimer's disease 5xFAD x Cx3cr1-Cre mice injected ICV with AAV9-flex-dCas9-KRAB-sgTYROBP at P60 Status: proposed

What This Experiment Tests

In_Vivo experiment designed to assess clinical efficacy targeting TYROBP in 5xFAD x Cx3cr1-Cre mice injected ICV with AAV9-flex-dCas9-KRAB-sgTYROBP at P60. Primary outcome: >=40% TYROBP knockdown in microglia; >=20% preservation of synaptic puncta at 6 months vs 5xFAD-vehi

Description

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.

TARGET GENE

TYROBP

MODEL SYSTEM

5xFAD x Cx3cr1-Cre mice injected ICV with AAV9-flex-dCas9-KRAB-sgTYROBP at P60

ESTIMATED COST

$95,000

TIMELINE

20 months

PATHWAY

TYROBP/DAP12 -> Syk/PLCg2 -> microglial activation -> synapse elimination

SOURCE

task:a989715e-c687-4558-91ca-74fce1474bd2

PRIMARY OUTCOME

>=40% TYROBP knockdown in microglia; >=20% preservation of synaptic puncta at 6 months vs 5xFAD-vehicle

Scoring Dimensions

Protocol

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.

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).

Related Hypotheses (1)

TYROBP (DAP12) Conditional Antagonism for Early-Stage Neuroprotection0.844

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