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
  • Base Sequence
  • Mutation/genetics
  • Fluorescence
  • Green Fluorescent Proteins/metabolism
  • Recombination, Genetic/genetics
  • Phenotype
  • Zebrafish/embryology
  • Zebrafish/genetics
  • Transcription Factors/metabolism
  • Animals
  • Transgenes
  • RNA, Guide, Kinetoplastida/genetics
  • Zebrafish Proteins/metabolism
  • Ribonucleoproteins/metabolism*
  • Genes, Reporter
  • Recombinant Fusion Proteins/metabolism
  • Multiprotein Complexes/metabolism*
  • CRISPR-Cas Systems/genetics*
  • Embryo, Nonmammalian/cytology
  • Embryo, Nonmammalian/drug effects
  • Embryo, Nonmammalian/metabolism
  • Solubility
  • Mutagenesis/genetics*
  • Binding Sites
  • Alleles
  • Morpholinos/pharmacology
(all 26)
PubMed
27130213 Full text @ Development
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
Figures
Figure Gallery (5 images)
Show all Figures
Expression
Phenotype
No data available
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
cz1701TgTransgenic Insertion
    cz1702TgTransgenic Insertion
      li1TgTransgenic Insertion
        1 - 3 of 3
        Show
        Human Disease / Model
        No data available
        Sequence Targeting Reagents
        Target Reagent Reagent Type
        amer1CRISPR1-amer1CRISPR
        amer1CRISPR2-amer1CRISPR
        atg7CRISPR1-atg7CRISPR
        becn1CRISPR1-becn1CRISPR
        camk2g1CRISPR1-camk2g1CRISPR
        ccn2aCRISPR1-ccn2aCRISPR
        gria3aCRISPR2-gria3aCRISPR
        hand2CRISPR1-hand2CRISPR
        hand2CRISPR2-hand2CRISPR
        kdm6alCRISPR1-kdm6alCRISPR
        1 - 10 of 29
        Show
        Fish
        No data available
        Antibodies
        Name Type Antigen Genes Isotypes Host Organism
        Ab3-tubamonoclonal
          		IgG1Mouse
          1 - 1 of 1
          Show
          Orthology
          No data available
          Engineered Foreign Genes
          Marker Marker Type Name
          CreEFGCre
          EGFPEFGEGFP
          mCherryEFGmCherry
          1 - 3 of 3
          Show
          Mapping
          No data available
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          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.