Research On Specific Types Of Neurons Labeled By Viral Strategy Based On CRISPR Combined With The Binary Expression System

The mammalian brain is composed of a large number of highly heterogeneous neurons,which are intertwined and connected to form complex neural circuits that process information and guide behaviors.Identifying the cell types of neurons is essential for studying neural circuits.Labeling neuronal types based on molecular markers is the preferred strategy for reliable experimental research on specific neuronal populations.Among them,the genetic strategies of relying on Cre strain mice to target specific types of neurons are commonly used labeling methods.However,the limited variety of Cre mice limits the development and application of such genetic strategies.In this thesis,a viral strategy combining the CRISPR-Cas9 system and modular binary expression system was developed.This strategy only needs to replace the AAV-g RNA(targeted gene)-driver virus,which can target neurons of different genotypes like Cre strain mice,thereby enriching the scope of tool selection of researchers.This strategy needs to inject three mixed viruses: AAV-g RNA(targeted gene)-driver,AAV-Sp Cas9,and AAVreporter.It uses CRISPR-Cas9-mediated homology-independent targeted integration(HITI)to integrate the ‘driver' gene into the specific location of the endogenous gene(targeted gene),to realize the cell-specific expression of the ‘driver' gene.The research results of this thesis are as follows.(1)In this research,four genes with different expression abundances in the mouse brain were selected,among which three genes(CaMKII?,Pthlh,Rxfp1)were designed as AAVg RNA(targeted gene)-tTA virus based on tTA/TRE binary system,and the fourth gene Gad1 was designed as AAV-g RNA(targeted gene)-Dre virus based on Dre/rox binary system,and three mixed viruses were injected into the brain regions where the corresponding genes were expressed.The results showed that different AAV-g RNA(targeted gene)-driver viruses designed for the two binary systems of the virus strategy could mark the neurons in the corresponding brain regions and their distal projections,and the marked projection areas were consistent with the results reported in the literature.It preliminarily proved that the viral strategy designed in this study is feasible.(2)To improve the accuracy of neuron type targeting,this thesis further combined the binary expression viral strategy with Cre strain mice,thus expanding an intersectional strategy targeting neuronal types defined by double genes.The intersectional strategy based on tTA/TRE needs to inject three mixed viruses: AAV-g RNA(targeted gene)-tTA,AAVSp Cas9,and AAV-TRE-DIO-reporter,while the intersectional strategy based on Dre/rox needs to inject three mixed viruses: AAV-g RNA(targeted gene)-Dre,AAV-DIO-Sp Cas9,and AAV-d DIO-reporter.Used this intersectional strategy,this thesis labeled CaMKII?/Drd1 neurons in the striatum,and CaMKII?/Drd1 neurons,CaMKII?/Sst neurons,and Gad1/Drd1 neurons in the primary motor cortex.The projections of CaMKII?/Drd1 neurons and CaMKII?/Sst neurons marked by the tTA/TRE intersectional strategy conform to the projection distribution of specific brain area neurons or Drd1 neuron type or Sst neuron type.The neurons labeled with another intersectional strategy based on Dre/rox had low brightness and no obvious axons.The above results of labeling neuronal types defined by double genes also showed that the viral strategy designed in this study is scalable and can reduce the use of double transgenic mice and even multiple transgenic mice.(3)This thesis verified that the insertion sequence in the CaMKII? neurons labeled by the tTA/TRE system can be correctly integrated into the CaMKII? site,and verified that the labeled neurons part belong to the CaMKII? neurons,and verified that the Gad1 neuron labeled by the Dre/rox system has an accuracy of 83.5%,which further illustrated the potential of the viral strategy to be popularized.