Large-scale F0 CRISPR screens in vivo using MIC-Drop.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
Jun 2023
Jun 2023
Historique:
received:
30
08
2022
accepted:
26
01
2023
medline:
12
6
2023
pubmed:
18
4
2023
entrez:
17
4
2023
Statut:
ppublish
Résumé
The zebrafish is a powerful model system for studying animal development, for modeling genetic diseases, and for large-scale in vivo functional genetics. Because of its ease of use and its high efficiency in targeted gene perturbation, CRISPR-Cas9 has recently gained prominence as the tool of choice for genetic manipulation in zebrafish. However, scaling up the technique for high-throughput in vivo functional genetics has been a challenge. We recently developed a method, Multiplexed Intermixed CRISPR Droplets (MIC-Drop), that makes large-scale CRISPR screening in zebrafish possible. Here, we outline the step-by-step protocol for performing functional genetic screens in zebrafish by using MIC-Drop. MIC-Drop uses multiplexed single-guide RNAs to generate biallelic mutations in injected zebrafish embryos, allowing genetic screens to be performed in F0 animals. Combining microfluidics and DNA barcoding enables simultaneous targeting of tens to hundreds of genes from a single injection needle, while also enabling retrospective and rapid identification of the genotype responsible for an observed phenotype. The primary target audiences for MIC-Drop are developmental biologists, zebrafish geneticists, and researchers interested in performing in vivo functional genetic screens in a vertebrate model system. MIC-Drop will also prove useful in the hands of chemical biologists seeking to identify targets of small molecules that cause phenotypic changes in zebrafish. By using MIC-Drop, a typical screen of 100 genes can be conducted within 2-3 weeks by a single user.
Identifiants
pubmed: 37069311
doi: 10.1038/s41596-023-00821-y
pii: 10.1038/s41596-023-00821-y
pmc: PMC10419324
mid: NIHMS1918458
doi:
Types de publication
Journal Article
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
1841-1865Subventions
Organisme : NHGRI NIH HHS
ID : K99 HG012593
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM134069
Pays : United States
Organisme : NHGRI NIH HHS
ID : T32 HG008962
Pays : United States
Informations de copyright
© 2023. Springer Nature Limited.
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