Lesson plan
Perturb-seq
Perturb-seq (also known as CRISP-seq and CROP-seq) refers to a high-throughput method of performing single cell RNA sequencing (scRNA-seq) on pooled genetic perturbation screens. Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation. Inferring a gene’s function by applying genetic perturbations to knock down or knock out a gene and studying the resulting phenotype is known as reverse genetics. Perturb-seq is a reverse genetics approach that allows for the investigation of phenotypes at the level of the transcriptome, to elucidate gene functions in many cells, in a massively parallel fashion. The Perturb-seq protocol uses CRISPR technology to inactivate specific genes and DNA barcoding of each guide RNA to allow for all perturbations to be pooled together and later deconvoluted, with assignment of each phenotype to a specific guide RNA. Droplet-based microfluidics platforms (or other cell sorting and separating techniques) are used to isolate individual cells, and then scRNA-seq is performed to generate gene expression profiles for each cell. Upon completion of the protocol, bioinformatics analyses are conducted to associate each specific cell and perturbation with a transcriptomic profile that characterizes the consequences of inactivating each gene.
Article summary
Perturb-seq (also known as CRISP-seq and CROP-seq) refers to a high-throughput method of performing single cell RNA sequencing (scRNA-seq) on pooled genetic perturbation screens. Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation. Inferring a gene’s function by applying genetic perturbations to knock down or knock out a gene and studying the resulting phenotype is known as reverse genetics. Perturb-seq is a reverse genetics approach that allows for the investigation of phenotypes at the level of the transcriptome, to elucidate gene functions in many cells, in a massively parallel fashion. The Perturb-seq protocol uses CRISPR technology to inactivate specific genes and DNA barcoding of each guide RNA to allow for all perturbations to be pooled together and later deconvoluted, with assignment of each phenotype to a specific guide RNA. Droplet-based microfluidics platforms (or other cell sorting and separating techniques) are used to isolate individual cells, and then scRNA-seq is performed to generate gene expression profiles for each cell. Upon completion of the protocol, bioinformatics analyses are conducted to associate each specific cell and perturbation with a transcriptomic profile that characterizes the consequences of inactivating each gene.
Key claims
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Discussion questions
- Summarise the article in your own words. Which single claim would you verify first, and why?
- Which claim in this article is most likely to be revised as the sources update? How would you check its current status?
- If two sources disagreed about one of these facts, what would you need in order to trust one over the other?
Bibliography
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Key claims and bibliography are generated from the same tracked claims and sources as the article page.