Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.
Autophagy
/ genetics
Autophagy-Related Proteins
/ genetics
CRISPR-Cas Systems
Carcinoma, Squamous Cell
/ mortality
Cell Proliferation
/ genetics
Cell Survival
/ genetics
Clustered Regularly Interspaced Short Palindromic Repeats
/ genetics
Databases, Genetic
Gene Knockout Techniques
/ methods
Gene Library
Gene Regulatory Networks
/ genetics
Genes, Essential
HEK293 Cells
High-Throughput Nucleotide Sequencing
Humans
Kaplan-Meier Estimate
Kelch-Like ECH-Associated Protein 1
/ genetics
Lung Neoplasms
/ mortality
Models, Genetic
RNA, Guide, Kinetoplastida
RNA-Seq
Vesicular Transport Proteins
/ genetics
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
04 06 2021
04 06 2021
Historique:
accepted:
14
04
2021
revised:
01
04
2021
received:
16
02
2021
pubmed:
7
5
2021
medline:
29
6
2021
entrez:
6
5
2021
Statut:
ppublish
Résumé
Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.
Identifiants
pubmed: 33956155
pii: 6270805
doi: 10.1093/nar/gkab309
pmc: PMC8191801
doi:
Substances chimiques
ATG2B protein, human
0
Autophagy-Related Proteins
0
KEAP1 protein, human
0
Kelch-Like ECH-Associated Protein 1
0
RNA, Guide
0
Vesicular Transport Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
5684-5704Informations de copyright
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.
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