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.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

“…The prevalence of other common deletions suggested that the NHEJ repair is not random. Inspection of the DSB-flanking sequences suggested that the most frequent alleles may result from microhomology-mediated repair ( Figure S1), supporting previous reports (Wang et al 2013;Thomas et al 2014).…”

Activity Suppression Behavior Phenotype in SULT4A1 Frameshift Mutant Zebrafish

“…mRNA for the left and right TALENs was generated in vitro and injected into type AB zebrafish larvae at the one-cell stage. This founder generation (F0) was raised to adulthood and screened for the presence of SULT4A1 mutations by HRMA (Parant et al, 2009;Dahlem et al, 2012;Thomas et al, 2014). Mutants in this founder generation were chimeric with the potential for multiple mutations.…”

“…TALENs were designed to target the zebrafish SULT4A1 gene. SULT4A1 gene exon sequences (GenBank accession number NP_001035334) were analyzed for potential TALEN targeting sites using the Old TALEN Targeter program at https://tale-nt.cac.cornell.edu/ node/add/talen-old (Cermak et al, 2011;Doyle et al, 2012;Thomas et al, 2014). TALEN targeting sites within the second exon were chosen with the following parameters: left target sequence, 59-ATTGATGAGCAGCTTCCAGT-39; left repeat array sequence, NI, NG, NG, NN, NI, NG, NN, NI, NN, HD, NI, NN, HD, NG, NG, HD, HD, NI, NN, NG; right target sequence, 59-AGCCGG-GATTGGAGATTATCC-39; right repeat array sequence, NN, NN, NI, NG, NI, NI, NG, HD, NG, HD, HD, NI, NI, NG, HD, HD, HD, NN, NN, HD; spacer length, 14 nucleotides.…”

“…Zebrafish represent an excellent model organism for the investigation of SULT4A1, due to the highly conserved nature of human SULT4A1 and zebrafish SULT4A1, which suggests an equally conserved function. Furthermore, recent advances in genomic editing technology have allowed researchers to quickly generate heritable gene-specific mutations in the zebrafish genome using transcription activator-like effector nucleases (TALENs) (Bedell et al, 2012;Dahlem et al, 2012;Thomas et al, 2014). In this work, we detail the generation and characterization of a strain of zebrafish containing an 8-nucleotide deletion (D8) in the SULT4A1 gene sequence that results in a frameshift mutation after 89 amino acids (AA) and premature stop codon after 132 AA.…”

Comparison of 3 Methodologies for Genotyping of Small Deletion and Insertion Polymorphisms

“…HRMA measures the decrease of fluorescence as double-stranded DNA denatures into single-stranded DNA, producing sequencespecific melting profiles that are dependent on amplicon lengths and GC content (18 -20 ). Because the 2 alleles of each DIP have substantial sequence differences, small biallelic DIPs are attractive candidates for genotyping by HRMA (13,21,22 ).…”