Sci Adv . . . The Wanyou/Yiming team accurately "deletes" rat-specific memories.

There are hundreds of different types of neurons in mammalian brain, which have different morphological, functional and genetic characteristics and are distributed in complex neural networks.different types of neurons in different neural circuits participate in different physiological and pathological phenotypes of mammals.to analyze the advanced cognitive function of the brain is inseparable from the gene manipulation of specific neural circuits and neurons.for example, our memory of events is encoded and stored by "imprinting cells" in the brain, and different memory is responsible for different groups of imprinted cells.traditional pharmacology or gene editing techniques can only affect a large number of neurons on a large scale and non selectively.is it possible for us to operate only a part of the imprinted cell population that we are interested in, so as to realize the precise manipulation of specific memory? On March 19, 2020, Wanyou and Yiming team, Institute of neuroscience, Peking University, published a research paper entitled "development of a crispr-sacas9 system for projection - and function specific gene editing in the rat brain" online in the journal Science advanced.based on CRISPR technology, combined with anterograde / retrograde AAV and activity-dependent cell labeling technology, this study realized the gene editing of pathway specific and function specific neuronal subpopulations, accurately controlled the storage and extinction of specific memory in rats, and provided a powerful strategy for analyzing the high-level cognitive function of the brain.with CBP (CREB binding protein) as the target gene, the researchers first verified the editing efficiency of crispr-sacas9 in vitro.sacas9 and gRNA were transfected into rat glioma cell lines by lentiviral vector. Western blot, immunofluorescence and T7 endonuclease assay showed that sacas9 could effectively knock down the expression of CBP protein.in vivo experiments, the anterior marginal cortex (PL) received direct projections from the dorsal hippocampal CA1 subregion (dca1) using anterograde trans synaptic AAV1 and retrograde rAAV2 retro.in order to specifically knock down the CBP gene of postsynaptic neurons, the study group injected AAV1 CRE into dca1 and expressed CRE dependent sacas9 in PL, successfully knocking down CBP gene of PL neurons receiving dca1 projection.after CBP gene knockdown, the expression of PSD-95 decreased, the density of dendritic spines decreased, and the excitability of neurons was inhibited.in the most interesting one-step experiment, the researchers induced fear memory of the experiment box in two different chambers (box a and Box B).furthermore, the CBP gene was specifically knocked down in the trace cells which stored fear memory in one of the experimental boxes (a box) by combining gene editing technology with activity-dependent cell marker technology.the results showed that this operation accurately "deleted" the fear memory of box a, while the memory of Box B remained intact.in addition, the study group specifically knocked down CBP gene of postsynaptic trace cells on dca1-pl pathway by using anterograde trans synaptic AAV1 and activity-dependent cell labeling technology, which successfully inhibited the long-term memory of rats, and proved that the dca1-pl pathway participated in the maintenance of long-term memory.in addition, the research group further realized gene editing of presynaptic neurons in the IL Amy (amygdala) pathway by using retrograde rAAV2 retro, which successfully inhibited the regression of fear memory in rats.finally, the research group verified the gene editing efficiency of sacas9 at the single cell level by using flow cytometry and high-throughput sequencing, and confirmed that sacas9 has a very low miss rate in neurons.in conclusion, crispr-sacas9, anterograde / retrograde AAV and activity-dependent cell labeling techniques were used to achieve pathway specific and functional specific gene editing in rat brain, and successfully controlled specific memory.there are many diseases, such as chronic pain, drug addiction, post-traumatic stress syndrome, etc., which are essentially "pathological memories" that are difficult to clear and exist for a long time after the patients have experienced the euphoria or pressure brought by pain and drugs. The specific mechanism is not completely clear, and there is no effective treatment.this memory editing system is expected to provide new ideas for the treatment of these diseases characterized by "pathological memory".SUN Haojie, a doctoral student from the Institute of neuroscience, Peking University, is the first author of this paper, and Professor Wanyou and researcher Yiming are the co authors.original link: plate maker: Ke