title: Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.
abstract: Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors: Linlin
authors: L
authors: Yin
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors: Lisette A
authors: LA
authors: Maddison
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors: Mingyu
authors: http://orcid.org/0000-0003-1217-2929
authors: M
authors: Li
authors: Department of Biological Science, Vanderbilt University, Nashville, Tennessee 37240.
authors: Nergis
authors: N
authors: Kara
authors: Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors: Matthew C
authors: http://orcid.org/0000-0001-9165-041X
authors: MC
authors: LaFave
authors: Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors: Gaurav K
authors: http://orcid.org/0000-0002-0429-1904
authors: GK
authors: Varshney
authors: Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors: Shawn M
authors: http://orcid.org/0000-0003-1147-0596
authors: SM
authors: Burgess
authors: Department of Biological Science, Vanderbilt University, Nashville, Tennessee 37240.
authors: James G
authors: http://orcid.org/0000-0001-5749-0870
authors: JG
authors: Patton
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 wenbiao.chen@vanderbilt.edu.
authors: Wenbiao
authors: http://orcid.org/0000-0002-0750-8275
authors: W
authors: Chen
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D015870:Gene Expression; D023061:Gene Order; D020868:Gene Silencing; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D017496:Hypopigmentation / Q000235:genetics; D016296:Mutagenesis; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D019076:Transgenes; D015027:Zebrafish
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D005947:Glucose
keywords: CRISPR/Cas9; conditional mutagenesis; glucose homeostasis; retinal regeneration; zebrafish