Menu

Massively Parallel Perturbations

Scientists combine CRISPR gene editing with single-cell sequencing for genotype-phenotype screens.

Mar 1, 2017
Ruth Williams

SINGLE-CELL SCREEN: A library of guide RNAs—each targeting a unique gene for CRISPR-based interference and carrying a unique barcode sequence—is introduced into a population of cells at a concentration that results in one guide RNA entering one cell, on average. Individual cells are then sorted into droplets bearing uniquely barcoded polyT primers, which are used to extract the cell’s mRNA. Sequencing the RNA then reveals both the introduced genetic mutations—determined by the guide RNA—and the transcriptional effect of that perturbation—determined by the collection of mRNAs bearing the cell-specific barcode (from the polyT primer).© GEORGE RETSECK

Determining how the genes in a cell affect its function is the overarching objective of molecular genetic studies. But most genotype-phenotype screens are limited by the number of genetic perturbations that can be feasibly measured in one experiment. In short, the more genetic disruptions examined, the more costly and time-consuming the experiments become.

Indeed, says Trey Ideker of the University of California, San Diego, very few large-scale genotype-phenotype screens have been performed, and those that have were mammoth undertakings. Now, thanks to two highly similar techniques—one called Perturb-Seq, developed by Aviv Regev of the Broad Institute and colleagues, and another, designed by Ido Amit of the Weizmann Institute in Israel and colleagues, called CRISP-Seq—it is possible to study numerous genetic manipulations, individually or combined, in thousands of single cells all in one experiment.

The principle behind Perturb-Seq and CRISP-Seq is to barcode both the individual genetic disturbances and the cells affected, such that sequencing can identify both. Briefly, a library of uniquely barcoded CRISPR guide RNAs targeting genes of interest is introduced into a population of cells. The mRNAs of individual cells are then extracted with uniquely barcoded primers. RNA sequencing reveals both the CRISPR-targeted gene (or genes) and the resulting transcriptional profile of the single cells. Importantly, tens of thousands of these cells can be sequenced in parallel.

Regev and Amit have used their techniques to examine, among other things, transcription factor functions and differentiation regulation in immune cells. But, says Ideker, the possibilities are endless. These are “the first models of this technology,” he says, “and they’re going to get better and better.” (Cell, 167:1853-66, 2016; Cell, 167:1867-82, 2016; Cell, 167:1883-96, 2016)

METHOD GENE DISRUPTION TRANSCRIPTOME ANALYSIS ESTIMATED COST* ESTIMATED TIME*
Gene knockout followed by transcriptome analysis Many options, including
gene editing (CRISPR), random mutagenesis, and
homologous recombination
 
Cells containing disrupted
genes are pooled and their mRNAs are extracted and sequenced.
$135,000 to analyze 1,000 gene perturbations by CRISPR 30 hours to analyze 1,000 gene perturbations by CRISPR

Perturb-Seq or CRISP-Seq

CRISPR/Cas9-driven
gene editing

Microfluidic technology isolates single cells carrying barcoded gene edits. The cells’ mRNAs are then extracted and sequenced with barcoded primers.

$40,000 to analyze 1,000 gene perturbations

12 hours to analyze 1,000 gene perturbations

*Estimations based on unpublished data from Aviv Regev and colleagues

 

April 2019

Will Car T Cells Smash Tumors?

New trials take the therapy beyond the blood

Marketplace

Sponsored Product Updates

Application of TruBIOME™ to Increase Mouse Model Reproducibility
Application of TruBIOME™ to Increase Mouse Model Reproducibility
With this application note from Taconic, learn about the effects of the microbiome on reproducibility and predictability and how TruBIOME™ helps researchers generate custom microbiota mouse models!
Getting More Consistent Results by Knowing the Quality of Your Protein
Getting More Consistent Results by Knowing the Quality of Your Protein
Download this guide from NanoTemper to learn how to identify and evaluate the quality of your protein samples!
Myth Busting: The Best Way to Use Pure Water in the Lab
Myth Busting: The Best Way to Use Pure Water in the Lab
Download this white paper from ELGA LabWater to learn about the role of pure water in the laboratory and the advantages of in-house water purification!
Shimadzu's New Nexera UHPLC Series with AI and IoT Enhancements Sets Industry Standard for Intelligence, Efficiency and Design
Shimadzu's New Nexera UHPLC Series with AI and IoT Enhancements Sets Industry Standard for Intelligence, Efficiency and Design
Shimadzu Corporation announces the release of the Nexera Ultra High-Performance Liquid Chromatograph series, incorporating artificial intelligence as Analytical Intelligence, allowing systems to detect and resolve issues automatically. The Nexera series makes lab management simple by integrating IoT and device networking, enabling users to easily review instrument status, optimize resource allocation, and achieve higher throughput.