PUBLICATION
Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes
- Authors
- Burger, A., Lindsay, H., Felker, A., Hess, C., Anders, C., Chiavacci, E., Zaugg, J., Weber, L.M., Catena, R., Jinek, M., Robinson, M.D., Mosimann, C.
- ID
- ZDB-PUB-160501-6
- Date
- 2016
- Source
- Development (Cambridge, England) 143(11): 2025-37 (Journal)
- Registered Authors
- Burger, Alexa, Chiavacci, Elena, Felker, Anastasia, Hess, Christopher, Mosimann, Christian
- Keywords
- CRISPR-Cas9, Zebrafish, Mutagenesis, Genome editing, RNP, CrispantCal, CrispRVariants
- MeSH Terms
-
- Alleles
- Animals
- Base Sequence
- Binding Sites
- CRISPR-Cas Systems/genetics*
- Embryo, Nonmammalian/cytology
- Embryo, Nonmammalian/drug effects
- Embryo, Nonmammalian/metabolism
- Fluorescence
- Genes, Reporter
- Green Fluorescent Proteins/metabolism
- Morpholinos/pharmacology
- Multiprotein Complexes/metabolism*
- Mutagenesis/genetics*
- Mutation/genetics
- Phenotype
- RNA, Guide, Kinetoplastida/genetics
- Recombinant Fusion Proteins/metabolism
- Recombination, Genetic/genetics
- Ribonucleoproteins/metabolism*
- Solubility
- Transcription Factors/metabolism
- Transgenes
- Zebrafish/embryology
- Zebrafish/genetics
- Zebrafish Proteins/metabolism
- PubMed
- 27130213 Full text @ Development
Citation
Burger, A., Lindsay, H., Felker, A., Hess, C., Anders, C., Chiavacci, E., Zaugg, J., Weber, L.M., Catena, R., Jinek, M., Robinson, M.D., Mosimann, C. (2016) Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development (Cambridge, England). 143(11):2025-37.
Abstract
CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro-assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene-regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping