Expression profiling by high throughput sequencing Other
Summary
The ever-growing compendium of genetic variants associated with human pathologies demands novel methods to study genotype-phenotype relationships in complex tissues in high-throughput. Here, we introduce AAV-mediated direct in vivo single-cell CRISPR screens, termed AAV-Perturb-seq, a tunable and broadly applicable method for 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 syndrome genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and novel 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 disease susceptibility genes important for dysfunctional RNA processing and synaptic function. Our work 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.
Overall design
Pooled screen data: A single dose of 5.0E+9 AAV particles carrying gRNAs to target 22q11.2 locus genes were injected intravenously into 8 weeks old male LSL-Cas9 mice. Animals were kept for four weeks under standard conditions before tissue extraction and processing. Arrayed confirmation experiments and CRISPRi experiment: Virus carrying gRNAs to target specific genes were individually prepared. Animals were split into cages accordingly to their experimental groups before tail vein injection of 5.0E+9 viral particles carrying unique gRNAs. Animals were kept for four weeks under standard conditions before tissue extraction and processing. For all experiments, animals were intravenously injected with a lethal dose of pentobarbital before transcardial perfusion with 15 mL of ice-cold PBS followed by 15 mL of ice-cold artificial cerebrospinal fluid. The brain was removed, sliced into 1 mm slices, and the region of interest was manually dissected. Nuclei isolation was performed with mechanical and chemical tissue dissociation procedures followed by FANS to isolate infected nuclei (pooled screen, arrayed and CRISPRi experiments) or purify nuclei from debris (LgDel model). Single cell libraries were generated using a modified version of the Chromium Single Cell 5สน Reagent Kit v1 (10x Genomics) to capture gene expression and gRNA information from each nucleus. Gene expression and gRNA libraries were sequenced with an Illumina NovaSeq 100 cycle kit.
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