Tracing The Expression Of Low-Abundant Transcripts And Lnc RNAs Using SPH-OminiCMV-Ents

The CRISPR/Cas system of bacterial and archaeal adaptive immunity is a kind of selfdefense mechanism to resist the invasion of pathogens and foreign nucleic acids.Due to its advantages of easy design,low cost,and high efficiency,it has been widely used in genome editing.Cas9 protein can be oriented to a specific site of the genome by sgRNA,inducing double strand break,thus achieving the gene knock-in and knock-out.When Cas9 mutates at two nuclease domains,it turns to a catalytically inactive Cas9(d Cas9)which holds the DNA binding activity only.When d Cas9 is fused to transcriptional effector or fluorescent protein,transcriptional activation/repression,epigenetic modification and imaging can be achieved.Many important progresses have been reported,but large size of the vector and low efficiency still limit its further application in vivo.Overexpression is still one of the most common method to upregulate target protein,and it is also a common method to encode a functional,therapeutic gene.However,the package size limits its application in Adeno-associated viruses(AAV).Currently,it can be packaged less than 5 kb of nucleic acids,limiting the overexpression for many genes.Thus,it is necessary to develop a more effective tool.CRISPR/d Cas9-based gene activation system have not been successfully used in vivo due to a lack of a more robust transcriptional activation system and the difficulty to deliver the large size vector.In this study,we optimized the existing activation system,established a platform named SPH,which induces an improved gene upregulation.Further we established a Cre-dependent SPH transgenic mouse and achieved the simultaneous transcriptional activation of multiple genes in vivo.Additionally,we also converted astrocyte to neuron by targeting three transcription factors,which provided a new platform for gene activation and treatments.The most commonly used method for detecting the activity of endogenous genes is fluorescent protein fusion strategy,however,it was difficult to detect genes with low expression level and lncRNAs.SPH activation system exhibits the ability to amplify gene expression,providing the possibility to trace endogenous transcriptional activity,particularly low-expressed genes and lncRNA? To explore it,we developed a broadspectrum endogenous transcription-gated switch(Ents),when it is coupled with SPHOmini CMV system,the detection of low-abundant genes and lncRNAs can be reliably achieved.Firstly,we developed a highly sensitive CRISPR-activator-associated reporter,named SPH-Omini CMV.Besides that,we identified tRNA as endogenous release mechanism to mature sgRNA which subesuqently triggered m Cherry expression and achieve the visualization of endogenous transcript.Moreover,SPH-Omini CMV-Ents visualized the dynamics of low-abundance transcripts and lncRNAs.It provides a powerful platform to detect the temporal and spatial expression of endogenous genes in living cells.In summary,our work has achieved simultaneous transcriptional activation of multiple genes in vivo,direct conversion of astrocyte to neuron in vivo,and established a new platform to detect the expression of low-expressed genes and lncRNA.