News

10

Massively Parallel Perturbations

posted on
Massively Parallel Perturbations

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 CRISPSeq— it is possible to study numerous genetic manipulations, individually or combined, in thousands of single cells all in one experiment.

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

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)

transcription factor functions and differentiation regulation in immune cells

 

| Categories: | Tags: CRISPR, gene editing, single-cell sequencing, genotype-phenotype screens | Comments: (0) | View Count: (243) | Return

Post a Comment