RESUMEN
Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.
Asunto(s)
Técnicas de Sustitución del Gen/métodos , Genómica/métodos , Ingeniería de Proteínas/métodos , Procesamiento Proteico-Postraduccional , Animales , Encéfalo/citología , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Dependovirus/genética , Edición Génica/métodos , Vectores Genéticos/genética , Humanos , Inmunoquímica/métodos , Inteínas , Ratones , Mutagénesis Insercional , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteómica , ARN Guía de Kinetoplastida/genética , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remain poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. Clustered regularly interspaced short palindromic repeats (CRISPR) depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced the frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders.