Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.
J Cell Biol
; 220(2)2021 02 01.
Article
in En
| MEDLINE
| ID: mdl-33464298
ABSTRACT
Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Genetic Testing
/
Genome
/
Imaging, Three-Dimensional
/
CRISPR-Cas Systems
Type of study:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limits:
Humans
Language:
En
Journal:
J Cell Biol
Year:
2021
Document type:
Article
Affiliation country:
Moldova
Publication country:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
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US
/
USA