title: Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.
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-sgRNA 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.
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Alexa
authors.initials: A
authors.lastname: Burger
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland SIB Swiss Institute of Bioinformatics, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Helen
authors.initials: H
authors.lastname: Lindsay
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Felker
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hess
authors.affiliation: Institute of Biochemistry, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Carolin
authors.initials: C
authors.lastname: Anders
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Elena
authors.initials: E
authors.lastname: Chiavacci
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Jonas
authors.initials: J
authors.lastname: Zaugg
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland SIB Swiss Institute of Bioinformatics, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Lukas M
authors.identifier: 0000-0002-3282-1730
authors.initials: LM
authors.lastname: Weber
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Raul
authors.initials: R
authors.lastname: Catena
authors.affiliation: Institute of Biochemistry, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Martin
authors.identifier: 0000-0002-7601-210X
authors.initials: M
authors.lastname: Jinek
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland SIB Swiss Institute of Bioinformatics, University of Zürich, Zürich 8057, Switzerland.
authors.forename: Mark D
authors.identifier: 0000-0002-3048-5518
authors.initials: MD
authors.lastname: Robinson
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland christian.mosimann@imls.uzh.ch.
authors.forename: Christian
authors.identifier: 0000-0002-0749-2576
authors.initials: C
authors.lastname: Mosimann
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D060172:Morpholinos / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D012261:Ribonucleoproteins / Q000378:metabolism*; D012995:Solubility; D014157:Transcription Factors / Q000378:metabolism; D019076:Transgenes; D015027:Zebrafish / Q000196:embryology / Q000235:genetics; D029961:Zebrafish Proteins / Q000378:metabolism
chemical_list: D060172:Morpholinos; D046912:Multiprotein Complexes; D000094704:RNA, Guide, CRISPR-Cas Systems; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: CRISPR-Cas9; CrispRVariants; CrispantCal; Genome editing; Mutagenesis; RNP; Zebrafish