PUBLICATION
CRIMP: a CRISPR/Cas9 insertional mutagenesis protocol and toolkit
- Authors
- Miles, L.B., Calcinotto, V., Oveissi, S., Serrano, R.J., Sonntag, C., Mulia, O., Lee, C., Bryson-Richardson, R.J.
- ID
- ZDB-PUB-240613-4
- Date
- 2024
- Source
- Nature communications 15: 50115011 (Journal)
- Registered Authors
- Bryson-Richardson, Robert, Calcinotto, Vanessa, Lee, Clara, Miles, Lee, Mulia, Orlen, Oveissi, Sara, Serrano, Rita, Sonntag, Carmen
- Keywords
- none
- MeSH Terms
-
- Alleles
- Animals
- CRISPR-Cas Systems*
- Gene Editing/methods
- Genetic Vectors/genetics
- Mutagenesis, Insertional*/methods
- Plasmids*/genetics
- Zebrafish*/genetics
- PubMed
- 38866742 Full text @ Nat. Commun.
Citation
Miles, L.B., Calcinotto, V., Oveissi, S., Serrano, R.J., Sonntag, C., Mulia, O., Lee, C., Bryson-Richardson, R.J. (2024) CRIMP: a CRISPR/Cas9 insertional mutagenesis protocol and toolkit. Nature communications. 15:50115011.
Abstract
Site-directed insertion is a powerful approach for generating mutant alleles, but low efficiency and the need for customisation for each target has limited its application. To overcome this, we developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination, generating null mutant alleles without inducing genetic compensation. The protocol results in a high frequency of integration events and can generate very early targeted insertions, during the first cell division, producing embryos with expression in one or both halves of the body plan. Fluorescent readout of integration events facilitates selection of successfully mutagenized fish and, subsequently, visual identification of heterozygous and mutant animals. Together, these advances greatly improve the efficacy of generating and studying mutant lines. The CRIMPkit contains 24 ready-to-use plasmid vectors to allow easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping