Science Advances . . . Peking University Wanyou/Yi Ming precision "delete" animal-specific memories.

Inature, based on CRISPR / cas9 genome editing technology, can effectively modify genes in various cell types (including neurons).however, even in the same brain region, the neuronal set is not uniform in anatomy or function, but is divided into different subgroups.this heterogeneity requires gene editing in a specific population of neurons.on March 20, 2020, Wanyou and Yiming, Institute of neuroscience, Peking University, jointly published a report entitled "development of a crispr-sacas9 system for projection - and function specific gene editing in the rat" online in science advances This study is based on CRISPR technology, combined with AAV and cell marker technology, to achieve gene editing in a functional specific mode, and to achieve precise deletion of specific memories in the brain of experimental rats.in conclusion, the crispr-sacas9 system, combined with electrophysiology, behavioral analysis, FACS and deep sequencing methods, provides a powerful strategy for precise genomic interference of brain function under physiological and pathological conditions.neurons with unique genetic, morphological and functional characteristics are organized into complex neural networks in mammalian brain.precise gene manipulation in specific neuronal subtypes and / or loops is essential to identify the causal relationship between neuronal activity and behavior.in vivo methods based on DNA antisense oligonucleotides and RNA interference have been widely used for gene silencing in the brain.however, it is still challenging to achieve stable gene knockout or gene modification in neuronal subpopulations with specific connectivity or functional characteristics, especially in rats and non-human primates.conditional recombination systems have been widely used to study brain function with spatiotemporal accuracy, but animal models may be labor-intensive and time-consuming, especially for transgenic rats.genome editing technology based on crispr-cas9 system can modify endogenous genes in various cell types quickly, efficiently and conveniently, resulting in frameshift insertion / deletion (indel) mutations and allow functional analysis of specific genes in the brain.more interestingly, in vivo gene editing can perform systematic genetic anatomy of neuronal circuits.however, even neuronal ensembles in the same brain region can be divided into different subgroups through anatomical afferent / efferent connections or functionally by recruitment of various tasks.this heterogeneity will need to be used to control interference techniques in specific neuronal populations, such as a specific crispr-cas9 method based on projection and function, to facilitate rapid gene editing in neural loop research.another obstacle in the application of crispr-cas9 system in nervous system is the limited load of virus vector.adeno associated virus (AAV) is one of the most commonly used vectors.the widely used endonuclease cas9 of Streptococcus pyogenes (spcas9) is limited by the capacity of general AAV delivery vectors (usually less than 4.4 to 4.7 KB) and low packaging efficiency. in contrast, the direct homologue of cas9 from Staphylococcus aureus (sacas9) was more than 1 KB shorter, but the efficiency of genome editing was similar to that of spcas9. in this study, the researchers aimed to develop a crispr-sacas9 system, which can edit target genes in neuronal subpopulations with specific connectivity or functional characteristics. as proof of concept, the researchers chose CBP (CREB binding protein) as the target gene, which is essential for neuronal excitability and memory formation, and tried to achieve the gene knockdown in the neural circuit related to fear memory with projection and function specificity. the high efficiency and specificity of crispr-sacas9 system can be widely used in the study of neural circuits. in conclusion, this study is the first application of CRISPR technology, which combines AAV and cell marker technology to realize gene editing in a function specific mode. the crispr-sacas9 system, combined with electrophysiology, behavioral analysis, FACS and deep sequencing methods, provides a powerful strategy for precise genomic interference of brain function under physiological and pathological conditions. reference message: