title: A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.
abstract: Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations inpycr1a,chd7andhace1genes. An insertion of seven nucleotides inpycr1aresulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion inchd7, suggesting activity of alternative translation sites in the other two mutants even thoughpycr1aexon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.
authors.affiliation: Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3K 6R8.
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Prykhozhij
authors.affiliation: Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3K 6R8.
authors.forename: Shelby L
authors.identifier: 0000-0001-8816-4752
authors.initials: SL
authors.lastname: Steele
authors.affiliation: Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3K 6R8.
authors.forename: Babak
authors.identifier: 0000-0001-6655-7563
authors.initials: B
authors.lastname: Razaghi
authors.affiliation: Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3K 6R8 jason.berman@iwk.nshealth.ca.|Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada B3H 4R2.|Department of Pathology, Dalhousie University, Halifax, NS, Canada B3H4R2.
authors.forename: Jason N
authors.identifier: 0000-0002-4053-6067
authors.initials: JN
authors.lastname: Berman
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics; D005453:Fluorescence; D016368:Frameshift Mutation / Q000235:genetics*; D017930:Genes, Reporter; D005818:Genetic Engineering; D005820:Genetic Testing / Q000379:methods*; D010641:Phenotype; D012326:RNA Splicing / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D029961:Zebrafish Proteins
keywords: CRISPR; Cas9; Mutation; Reporter; SgRNA; Zebrafish