|
Research Purpose |
Expression box core |
Primary Risks |
|
Overexpression/Functional Rescue |
Coding sequence + adapter promoter |
Overexpression toxicity, non-target tissue expression |
|
RNA intervention |
shRNA/miRNA or regulatory RNA |
Dose-dependent toxicity, off-target and processing saturation |
|
Gene editing |
Nuclease/Editor + gRNA; double AAV if necessary |
Capacity, immunity, off-target, long-term nuclease expression |
|
Neural circuit tracing |
fluorescence/transsynaptic or retrograde tools |
Directivity, leakage, toxicity and time windows |
|
Sensing and Control |
Sensors, DREADD/optogenetic components |
Expression level affects signal-to-noise ratio and physiological status |
The selection sequence should first determine "what the cell needs to do" and then decide on the capsid. The capsid mainly affects arrival and entry, and the promoter/enhancer determines the expression profile after entry, and the two cannot replace each other. Editing and tracing projects should also set up independent controls for the vector itself, effectors, and readout systems. [9-1 to 9-3]
References for this chapter
[9-1] Wang D, Zhang F, Gao G. CRISPR-based therapeutic genome editing: strategies and in vivo delivery by AAV vectors. Cell. 2020;181:136-150. DOI:10.1016/j.cell.2020.03.023. PMID:32243786. [H]
[9-2] Tervo DGR, et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron. 2016;92:372-382. DOI:10.1016/j.neuron.2016.09.021. PMID:27720486. [H]
[9-3] Davis JR, et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat Biotechnol. 2024;42:253-264. DOI:10.1038/s41587-023-01758-z. [N]