PUBLICATION
High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products
- Authors
- Colijn, S., Yin, Y., Stratman, A.N.
- ID
- ZDB-PUB-220213-1
- Date
- 2022
- Source
- Developmental Biology 484: 22-29 (Journal)
- Registered Authors
- Stratman, Amber
- Keywords
- CRISPR, Cilia, Fragment analyzer, Genotyping, Indels, Zebrafish
- MeSH Terms
-
- Zebrafish*/genetics
- Polymerase Chain Reaction
- Clustered Regularly Interspaced Short Palindromic Repeats*
- CRISPR-Cas Systems/genetics
- Animals
- Caenorhabditis elegans/genetics
- PubMed
- 35149003 Full text @ Dev. Biol.
Citation
Colijn, S., Yin, Y., Stratman, A.N. (2022) High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products. Developmental Biology. 484:22-29.
Abstract
Targeted mutagenesis in zebrafish, fruit flies, and C. elegans has been significantly improved over the years through CRISPR technology. CRISPR enables researchers to efficiently examine cellular pathways by inducing small, targeted mutations in vivo. Though these mutations are commonly random insertions or deletions (indels), they often result in functionally disrupted alleles of a target gene if the CRISPR components are appropriately designed. However, current protocols used to identify the presence of CRISPR-generated indels are often labor intensive, time-consuming, or expensive. Here, we describe a straightforward, high-throughput method for identifying the presence of mutations by using a fragment analyzer platform which allows for DNA fragment sizing through high-resolution capillary gel-electrophoresis. Following this protocol, small indels-down to 2 base pairs-can be quickly and reliably identified, thus allowing for large-scale genotyping of newly-generated or stable mutant lines.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping