Perturb-seq

Perturb-seq is a technique in which single-cell RNA sequencing is used to measure the effects of many CRISPR-based perturbations on large numbers of cells. The method was developed by researchers at UC San Francisco and the Broad Institute of MIT and Harvard. To identify specific DNA changes or perturbations on individual cells within pooled experiments, cell barcoding is used on single cells from which transcriptional profiles were also acquired. Using droplet-based single-cell RNA sequencing, each cell has a gene expression profile associated with one or more genetic perturbations. Since Perturb-seq analyzes single cells, researchers can study complex phenotypes that may be missed when the response of groups of cells is averaged.

Perturb-Seq is a single-cell CRISPR screening (scCRISPR) technique that uses RNA-seq to uncover genotype-phenotype relationships at the transcriptome level. Gene function is perturbed by the introduction of single guide RNA (sgRNA) delivered to cells. sgRNA is an engineered version of the two-piece guide RNA complex with a portion that is complementary to the desired target DNA sequence and functions to bring the CRISPR-associated (Cas) protein, Cas9, to the site. Perturb-seq is compatible with several CRISPR-based perturbations such as CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and CRISPR knockout. CRISPRi represses the expression of target genes by interfering with transcriptional elongation. In screening experiments, libraries containing hundreds to thousands of sgRNAs with different targets are introduced into cells at a concentration such that each cell receives one sgRNA.

In 2016, two initial Perturb-seq papers were published in Cell. Researchers of the paper led by the Broad Institute of MIT and Harvard used CRISPR-Cas9 nucleases to cut DNA and inactivate genes for transcription factors (TFs), proteins that control the expression of genes involved in immune response in dendritic cells. They also inactivated genes for TFs and cell cycle regulator proteins in a cancer cell line. Lead author Aviv Regev is a scientific advisor to ThermoFisher, Syros, and Driver.

UC-San Francisco-led experiments used CRISPR-based transcriptional interference (CRISPRi) to repress genes in a cancer cell line in combination with Perturb-seq to investigate errors in protein production that result from the cell sensing stress. The experiments interrogate the functional relationships between genes involved in endoplasmic reticulum (ER) homeostasis and unfolded protein response (UPR). UPR is an integrated ER stress-response pathway in which various cell perturbations are sensed and cause activation of transcription factors that promote survival or trigger cell death. Lead author Jonathan S. Weissman is founder of KSQ Therapeutics, a company that uses CRISPR for genome screening.

Perturb-Seq was used to study the effects of knocking out thirty-five candidate genes for autism spectrum disorders (ASDs) in mice in research co-led by Aviva Regev and MIT researcher Feng Zhang.

Perturb-seq was first used at genome-scale in 2022 by a group co-led by Weissman and Thomas M. Norman at Sloan Kettering Institute, New York. All expressed genes were targeted with CRISPRi across more than 2.5 million human cells. The resulting transcriptional profiles were used to predict gene function and identify genes involved in complex cellular phenomena. The researchers used two cell lines: chronic myeloid leukemia (CML) (K562) and retinal pigment epithelial (RPE1).

Perturb-Seq was applied to cancer to assess the potential impact of cancer gene variants on tumor behavior. The impact of 200 TP53 and KRAS variants on RNA profiles was assessed in over 300,000 single lung cancer cells and categorized depending on whether the phenotype was gain-of-function, loss-of-function, or dominant negative.