title: High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.
abstract: With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Holly R
authors.initials: HR
authors.lastname: Thomas
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Stefanie M
authors.initials: SM
authors.lastname: Percival
authors.affiliation: Department of Cellular, Developmental, and Integrated Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Bradley K
authors.initials: BK
authors.lastname: Yoder
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: John M
authors.initials: JM
authors.lastname: Parant
mesh_terms: D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004249:DNA Damage; D004252:DNA Mutational Analysis; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D023281:Genomics; D060005:Genotyping Techniques / Q000379:methods*; D006824:Hybridization, Genetic; D009154:Mutation; D009691:Nucleic Acid Denaturation; D010641:Phenotype; D044366:Transition Temperature; D015027:Zebrafish / Q000235:genetics*; D016335:Zinc Fingers
chemical_list: D003851:Deoxyribonucleases