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      Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility

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          Abstract

          Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ ercc4, fanct/ ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp -/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

          Author summary

          Deficiencies in repair of DNA damage can cause diseases such as Fanconi anemia (FA), which is characterized by birth defects, bone marrow failure, anemia, leukemia and other cancers. A set of proteins constitute the FA pathway and together orchestrate the DNA repair process. Inactivation of one or more gene(s) encoding the proteins of the DNA repair pathway in an animal model would enable us to study the functions of these proteins in maintenance of normal cellular functions and the overall health of an individual in the absence of function. We systematically targeted the FA pathway in zebrafish using CRISPR/Cas9. We generated 36 fish lines with loss-of-function mutations in 19 FA pathway genes and showed that all survive to adulthood. We did not notice obvious morphological changes except in fancp gene-inactivated fish, which were smaller in length. However, all mutant fish were either exclusively or in majority male. Unlike reduced fertility among FA patients, all adult mutant fish were fertile, except for the fancd1 and fancj knockout males. These mutant zebrafish will serve as a huge resource for the scientific community to study the role of FA proteins in fish development, DNA repair, and as models for FA disease.

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          Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

          A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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            Genetic compensation: A phenomenon in search of mechanisms

            Several recent studies in a number of model systems including zebrafish, Arabidopsis, and mouse have revealed phenotypic differences between knockouts (i.e., mutants) and knockdowns (e.g., antisense-treated animals). These differences have been attributed to a number of reasons including off-target effects of the antisense reagents. An alternative explanation was recently proposed based on a zebrafish study reporting that genetic compensation was observed in egfl7 mutant but not knockdown animals. Dosage compensation was first reported in Drosophila in 1932, and genetic compensation in response to a gene knockout was first reported in yeast in 1969. Since then, genetic compensation has been documented many times in a number of model organisms; however, our understanding of the underlying molecular mechanisms remains limited. In this review, we revisit studies reporting genetic compensation in higher eukaryotes and outline possible molecular mechanisms, which may include both transcriptional and posttranscriptional processes.
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              High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

              The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F 1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Methodology
                Role: Methodology
                Role: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: Methodology
                Role: Methodology
                Role: Data curationRole: Methodology
                Role: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                12 December 2018
                December 2018
                : 14
                : 12
                : e1007821
                Affiliations
                [1 ] Cancer Genomics Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
                [2 ] Zebrafish Core, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
                [3 ] Genomics Core, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America
                Stanford University School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                ‡ These authors also contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-1672-1887
                http://orcid.org/0000-0002-4804-5676
                http://orcid.org/0000-0003-2571-4803
                http://orcid.org/0000-0001-5487-1766
                http://orcid.org/0000-0001-8330-1279
                http://orcid.org/0000-0002-7668-4723
                http://orcid.org/0000-0001-5565-662X
                http://orcid.org/0000-0002-8084-0530
                Article
                PGENETICS-D-18-00586
                10.1371/journal.pgen.1007821
                6328202
                30540754
                97e268b9-bdc4-4404-a71b-78990e9f1050

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 21 March 2018
                : 8 November 2018
                Page count
                Figures: 7, Tables: 1, Pages: 27
                Funding
                Funded by: The intramural research program of the National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland
                Award Recipient :
                This work was supported by the intramural research program of the National Human Genome Research Institute, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Zebrafish
                Research and Analysis Methods
                Model Organisms
                Zebrafish
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Animal Models
                Zebrafish
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Fish
                Osteichthyes
                Zebrafish
                Biology and Life Sciences
                Developmental Biology
                Embryology
                Embryos
                Biology and Life Sciences
                Genetics
                Genetic Loci
                Alleles
                Biology and Life Sciences
                Genetics
                Mutation
                Frameshift Mutation
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Reverse Transcriptase-Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Reverse Transcriptase-Polymerase Chain Reaction
                Biology and Life Sciences
                Genetics
                Phenotypes
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Genetic Recombination
                Gene Knockout
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Genetic Recombination
                Gene Knockout
                Biology and Life Sciences
                Genetics
                Mutation
                Custom metadata
                vor-update-to-uncorrected-proof
                2019-01-10
                All relevant data are within the paper and its Supporting Information files.

                Genetics
                Genetics

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