title: A simple and efficient method to visualize and quantify the efficiency of chromosomal mutations from genome editing.
abstract: Genome editing with designer nucleases such as TALEN and CRISPR/Cas enzymes has broad applications. Delivery of these designer nucleases into organisms induces various genetic mutations including deletions, insertions and nucleotide substitutions. Characterizing those mutations is critical for evaluating the efficacy and specificity of targeted genome editing. While a number of methods have been developed to identify the mutations, none other than sequencing allows the identification of the most desired mutations, i.e., out-of-frame insertions/deletions that disrupt genes. Here we report a simple and efficient method to visualize and quantify the efficiency of genomic mutations induced by genome-editing. Our approach is based on the expression of a two-color fusion protein in a vector that allows the insertion of the edited region in the genome in between the two color moieties. We show that our approach not only easily identifies developing animals with desired mutations but also efficiently quantifies the mutation rate in vivo. Furthermore, by using LacZα and GFP as the color moieties, our approach can even eliminate the need for a fluorescent microscope, allowing the analysis with simple bright field visualization. Such an approach will greatly simplify the screen for effective genome-editing enzymes and identify the desired mutant cells/animals.
authors.affiliation: Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 18 Library Dr., Bethesda, Maryland, 20892, United States.
authors.forename: Liezhen
authors.initials: L
authors.lastname: Fu
authors.affiliation: Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 18 Library Dr., Bethesda, Maryland, 20892, United States.
authors.forename: Luan
authors.initials: L
authors.lastname: Wen
authors.affiliation: Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 18 Library Dr., Bethesda, Maryland, 20892, United States.
authors.forename: Nga
authors.initials: N
authors.lastname: Luu
authors.affiliation: Section on Molecular Morphogenesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 18 Library Dr., Bethesda, Maryland, 20892, United States.
authors.forename: Yun-Bo
authors.initials: YB
authors.lastname: Shi
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002869:Chromosome Aberrations; D000072669:Gene Editing; D023061:Gene Order; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D023281:Genomics / Q000379:methods; D009154:Mutation; D011993:Recombinant Fusion Proteins; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
chemical_list: D011993:Recombinant Fusion Proteins; D000069896:Transcription Activator-Like Effector Nucleases