ZFIN ID: ZDB-PUB-160501-6
Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes
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.
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/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
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.