PUBLICATION
Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish
- Authors
- Klatt Shaw, D., Mokalled, M.H.
- ID
- ZDB-PUB-210321-7
- Date
- 2021
- Source
- G3 (Bethesda) 11(8): (Journal)
- Registered Authors
- Mokalled, Mayssa
- Keywords
- CRISPR/Cas9, Spinal cord injury, genetic screen, regeneration, zebrafish
- MeSH Terms
-
- Phenotype
- Mutagenesis
- Animals
- INDEL Mutation
- CRISPR-Cas Systems*
- PubMed
- 33742663 Full text @ G3 (Bethesda)
Abstract
Adult zebrafish are increasingly used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 'crispants'. We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.
Genes / Markers
Figure Gallery (6 images)
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping