MIC-Drop: A platform for large-scale in vivo CRISPR screens

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a pla...

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Published inScience (American Association for the Advancement of Science) Vol. 373; no. 6559; pp. 1146 - 1151
Main Authors Parvez, Saba, Herdman, Chelsea, Beerens, Manu, Chakraborti, Korak, Harmer, Zachary P, Yeh, Jing-Ruey J, MacRae, Calum A, Yost, H Joseph, Peterson, Randall T
Format Journal Article
LanguageEnglish
Published United States The American Association for the Advancement of Science 03.09.2021
Subjects
Animals
Cardiovascular System - growth & development
Cell Culture Techniques
CRISPR
CRISPR-Cas Systems
Danio rerio
Deoxyribonucleic acid
DNA
Droplets
Gene sequencing
Genes
Genetic screening
Genetic Testing
Genetics
Genomes
High-Throughput Nucleotide Sequencing
Microfluidic Analytical Techniques
Microfluidics
Multiplexing
Mutants
Organisms
Phenotypes
Target recognition
Zebrafish
Zebrafish - genetics
Zebrafish - growth & development
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Summary:CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we showed that MIC-Drop could identify small-molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discovered several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse genetic screens in model organisms.
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Author contributions: S.P. and R.T.P contributed to conception and design, collection, analysis, and interpretation of the data, and manuscript writing. J. J. Y. contributed to conceptualization and experimental design. C.H. and H.J.Y. performed RNAseq data analysis. M.B. and C.A.M. performed and interpreted cardiac voltage mapping experiments. K.C. and Z.P.H. provided help with testing and optimizing the MIC-Drop platform. All authors contributed to the writing and review of the manuscript.
Current address: Cellular and Molecular Biology, University of Wisconsin-Madison, WI, USA.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.abi8870