PUBLICATION
Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish
- Authors
- Voigt, B., Minowa, R., Gray, R.S.
- ID
- ZDB-PUB-230228-40
- Date
- 2023
- Source
- Journal of visualized experiments : JoVE (192): (Journal)
- Registered Authors
- Gray, Ryan, Minowa, Ryoko
- Keywords
- none
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems
- Endonucleases/genetics
- Gene Editing/methods
- Germ Cells/metabolism
- Male
- Semen*/metabolism
- Spermatozoa/metabolism
- Zebrafish*/genetics
- Zebrafish*/metabolism
- PubMed
- 36847371 Full text @ J. Vis. Exp.
Citation
Voigt, B., Minowa, R., Gray, R.S. (2023) Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish. Journal of visualized experiments : JoVE. (192):.
Abstract
The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping