CRISPR-Map:A Labeling Technology For Specific Neuron Subtypes

The human brain is an extremely complex structure that not only has nearly 100 billion neurons,but also contains many types of neurons.Different types of neurons have different biological characteristics and perform different functions.However,their distribution in the brain is mixed and difficult to distinguish from each other.This is a big challenge for brain research.Cell-type specific labeling of neurons is a necessary prerequisite for analyzing complex brain structure and brain function.However,traditional labeling approaches relying on Credriver transgenic mice can only label a large class of neurons expressing the corresponding genotypes,and it is still difficult to accurately distinguish between different subtypes of neurons.In order to solve this problem,this paper established a CRISPR/Cas9-mediated recombinase integration system,which we called CRISPR-Map system.Combining with Cre-driver transgenic mice,this system can achieve accurate labeling of specific neuron subtype.The main research contents of the study are listed below:(1)In order to screen vector tools with rigorous expression in CRISPR-Map system,three Dre donor plasmid vectors with different structures were constructed,and through Comparative experiments,we found the Dre donor plasmid p L-stop-i Dre-g RNA and its packaged viral vector AAV-Stop-i Dre-g RNA have high rigor and can be potentially used as a labeling system for this study.(2)CRISPR-Map system was tested by labeling the inhibitory subtypes of dopamine receptor D1 expressing medium spiny neurons(Drd1+/Gad1+ neurons)in the caudateputamen(Cpu)region of mice.Experimental results confirmed that the CRISPR-Map system can be used to specifically label specific subtypes of neuron.(3)We achieved the sparse labeling of Drd1+/Gad1+ neurons by reducing the titer of Dre donor virus using the CRISPR-Map labeling system.Moreover,two subtypes of Sst intermediate neurons(Chod1 and Esm1)were labeled by the this system.The experimental results showed that the structure of the neurons labeled by the marker system was clear and suitable for further study of the single neuron structure of the subtype of specific type of neurons.In summary,we established a CRISPR / Cas9-mediated recombinase integration system,CRISPR-Map system,which can specifically label specific subtypes of neurons.The labeling system can greatly reduce the dependence on transgenic mice and is a good supplement to the existing neuron labeling methods,and lays a solid foundation for further analysis of the structure and function of specific neuronal subtypes in the future.