PUBLICATION
Large-scale F0 CRISPR screens in vivo using MIC-Drop
- Authors
- Parvez, S., Brandt, Z.J., Peterson, R.T.
- ID
- ZDB-PUB-230418-65
- Date
- 2023
- Source
- Nature Protocols 18(6): 1841-1865 (Review)
- Registered Authors
- Peterson, Randall
- Keywords
- none
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*/genetics
- Genetic Testing
- Phenotype
- Retrospective Studies
- Zebrafish*/genetics
- PubMed
- 37069311 Full text @ Nat. Protoc.
Citation
Parvez, S., Brandt, Z.J., Peterson, R.T. (2023) Large-scale F0 CRISPR screens in vivo using MIC-Drop. Nature Protocols. 18(6):1841-1865.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping