Efficient genome editing in the mouse brain by local delivery of engineered Cas9 RNP complexes

Link to paper

Summary

1. The study demonstrates efficient genome editing in post-mitotic neurons of the adult mouse brain using Cas9 ribonucleoprotein (RNP) complexes → holds promise for correcting/inactivating genetic causes of neurological diseases characterized by specific neuronal populations via safe Cas9 delivery with minimal immune response
2. Engineered Cas9 variants with multiple SV40 nuclear localization sequences (NLS) enhance the efficiency of neuronal genome editing in vivo by 10x, addressing the challenge of tissue-specific delivery
3. Mouse model Ai9 tdTomato is used to detect Cas9 editing, providing a quantitative method for genome modification. Specific guide RNAs activate tdTomato expression in genetically modified cells
4. Increasing the number of NLS sequences on Cas9 improves cell penetration and editing efficiency in cultured cells. Mixing RNPs with Lipofectamine2000 (transfection reagent) also facilitates RNP delivery into the cytoplasm in various cell types

Other Notes

Background

what is the gap in knowledge this research is trying to address?

• Previous studies have shown the utility of Cas9-mediated genome editing in various tissues, but the challenge of tissue-specific delivery, especially in the complex neural environment, remain a significant hurdle

Hypothesis

The engineered Cas9 RNP complexes with multiple NLS will enhance the efficiency of genome editing in neurons within the adult mouse brain. - These RNPs can overcome issues related to tissue-specific delivery, immune responses, and off-target effects, making them a more promising tool for correcting genetic causes of neurological diseases.

Experiments

1. Testing different Cas9 RNP variants with varying NLS sequences in cultured cells and in the adult mouse brain
2. Stereotaxic injections of RNPs targeting tdTomato in various brain regions and subsequent analysis of edited cells
3. Dual-color immunofluorescence to identify the specific neuronal subtypes edited by 4xNLS-Cas9-2xNLS RNPs
4. Dose-dependent experiments to assess the effect of RNP quantity on the number and volume of edited cells
5. Analysis of microglia activation and immune response in the brain following RNP injection

Observations and Conclusions

1. Engineered Cas9 RNP complexes, particularly the 4×NLS-Cas9-2×NLS variant, demonstrate a tenfold increase in efficiency for genome editing in neurons within the adult mouse brain, effectively addressing the challenge of tissue-specific delivery
   • i.e. NLS-rich CRISPR enzymes can self-deliver into neurons following local injection into brain tissue
   • NLS signals are similar to a typical cell-penetrating peptide
2. In vivo genome editing with these RNPs is specific to the target gene (tdTomato), showing that RNPs are precise and do not result in off-target effects
3. The RNP-mediated editing is observed in various neuronal subtypes within the brain, confirming the potential for neuron-specific targeting and treatment of genetic neurological diseases
4. Increasing the RNP dose leads to a higher number and volume of genome-edited cells, indicating a dose-dependent response to RNP delivery
5. The study shows minimal immune response to Cas9 RNPs injected into the brain, suggesting the feasibility of safe and precise Cas9-RNP delivery for therapeutic purposes

Iteration / Future Directions

1. Further exploration of the mechanisms underlying the in vivo neurotropism of 4×NLS-Cas9-2×NLS RNPs to understand how they specifically target neurons, potentially leading to the development of even more efficient delivery methods
2. Additional research to assess the safety and potential adaptive responses in the context of therapeutic Cas9-RNP delivery, with a focus on minimizing any immune-related side effects