Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs

Abstract

Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.

Figure 1.

Targeting efficiencies and toxicities of homodimeric and heterodimeric TALENs in zebrafish. (a) Percentages of dead, deformed and normal (OK) embryos at 1 day after injection with TALEN-coding mRNAs. The total number of embryos scored (n) is 165, 110 and 127 for WT, EL/KK and ELD/KKR, respectively. (b) Mutation rates in somatic zebrafish cells are determined as described in ‘Materials and Methods’ section and are shown here. Mutated sequences can be found in Supplementary Figure S5. The TALENs constructed in two heterodimeric frameworks showed increased targeting efficiencies and reduced toxicities compared to their homodimeric counterparts.

Figure 2.

Engineered TALENs are highly efficient in targeting endogenous zebrafish genes. (a) Frequencies of mutations induced by homodimeric and heterodimeric TALENs targeting 10 endogenous loci in zebrafish. The number of clones depicts the total number of alleles including wild-type and mutant alleles that have been sequenced. The ratios of mutation rates between heterodimeric and homodimeric TALENs are also shown. (b) Comparison of the toxicity induced by homodimeric and heterodimeric TALENs. The percentages of normal embryos at 1 day after injection with TALEN-coding mRNAs are shown. A total of 30–189 embryos were scored for each TALEN pair. The number of embryos scored for each TALEN pair is shown in Supplementary Figure S8.

Figure 3.

Frequencies and sequences of somatic mutations induced by TALENs at their intended or variant target loci. Both homodimeric and heterodimeric TALENs targeting epas1b.2 induced high rates of indel mutations at their intended target loci. However, their targeting efficiencies at a variant target locus with a 2 base pair mismatch were reduced significantly. The TALEN constructs used and their mutation rates are as indicated (SQT418/419, homodimeric TALEN constructs; WH7/8, ELD/KKR heterodimeric TALEN constructs). The wild-type sequence is shown at the top with TALEN binding sites marked in yellow. Deletions are indicated by gray highlighted red dashes and insertions by blue highlighted lower case letters. The sizes of the indels are labeled to the right of each sequence (+, insertion; −, deletion). The number of times each mutant allele was isolated is shown in brackets.

Figure 4.

TALENs induce high rates of heritable mutations. Fish that had been previously injected with either homodimeric or heterodimeric TALENs were screened for founders carrying heritable mutations. The targeted gene and the TALEN pairs injected are indicated at the top of each panel. Individual embryos from each potential founder were lysed for PCR analysis. Indel mutations were identified by the presence of the PCR size variants. Except for hey2 and gria3a, the sequences of the mutant alleles have been determined by sequencing (Figure 5). TALEN 1295/1260, 1297/1257 and the SQT constructs encode homodimeric TALEN pairs, whereas the WH constructs encode ELD/KKR heterodimeric TALENs. ^aThe number of embryos that possessed indel mutations are shown outside of the parentheses, and the sizes of the indels are shown inside the parentheses. Some of the mutation sequences are shown in Figure 5.

Figure 5.

Sequences of heritable mutations induced by TALENs. For six of the eight genes that are shown in Figure 4, we have determined the sequences of all the mutant alleles identified in the progeny of the founders by sequencing. Potential founders were generated by injection of either homodimeric (SQT constructs) or heterodimeric (WH constructs) TALEN mRNAs as indicated. The MT numbers shown at the left of the sequences represent the founder numbers, which correspond to the founder numbers shown in Figure 4. Sometimes different mutant alleles were found in different embryos from a single founder. For the sequences, the wild-type sequence is shown at the top with TALEN binding sites marked in yellow. Deletions are indicated by gray highlighted red dashes and insertions by blue highlighted lower case letters. The sizes of the indels are labeled to the right of each sequence (+, insertion; −, deletion).