Pooled genetic perturbation screens with image-based phenotypes
Name
nihms-1846522.pdf
Description
Published version
Size
2.11 MB
Format
Adobe PDF
Checksum (MD5)
0fa9d449b9e325854c190424be19531a
Author(s)
Feldman, David
Funk, Luke
Le, Anna
Carlson, Rebecca J
Leiken, Michael D
Tsai, FuNien
Soong, Brian
Singh, Avtar
Blainey, Paul C
Date Issued
2022
Journal
Nature Protocols
Publisher
Springer Science and Business Media LLC
Citation
Feldman, David, Funk, Luke, Le, Anna, Carlson, Rebecca J, Leiken, Michael D et al. 2022. "Pooled genetic perturbation screens with image-based phenotypes." Nature Protocols, 17 (2).
Version
Final published version
Abstract
Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >106 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Creative Commons Attribution 4.0 International license
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1038/S41596-021-00653-8
