RESUMEN
The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner1,2. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome3,4 genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.
Asunto(s)
Sistemas CRISPR-Cas , Dependovirus , Edición Génica , Estudios de Asociación Genética , Análisis de la Célula Individual , Transcripción Genética , Animales , Humanos , Ratones , Dependovirus/genética , Estudios de Asociación Genética/métodos , Neuronas/metabolismo , Fenotipo , Corteza Prefrontal/metabolismo , Transcripción Genética/genética , Análisis de la Célula Individual/métodos , Sistemas CRISPR-Cas/genética , Síndrome de DiGeorge/tratamiento farmacológico , Síndrome de DiGeorge/genética , Modelos Animales de Enfermedad , Procesamiento Postranscripcional del ARN , Sinapsis/patología , Predisposición Genética a la EnfermedadRESUMEN
The genomes of Lactobacillus curvatus KG6, L. curvatus MRS6, and Lactobacillus sakei FAM18311 were sequenced and assembled using PacBio single-molecule real-time (SMRT) technology. The strains were isolated from Swiss fermented meat products. Circular chromosomes were of 1.98 Mbp (KG6), 2.11 Mbp (MRS6), and 1.95 Mbp (FAM18311), with a G+C content of 41.3 to 42.0%.