TALENs have been shown previously to be capable of generating mutations in endogenous zebrafish genes (19
), but success rates, somatic mutation rates, toxicities and germline transmission efficiencies determined from large-scale studies have not yet been reported. In addition, the most recent generation of obligate heterodimeric FokI nuclease domains have not yet been used with TALENs in zebrafish (24
). In this study, we sought to use the FLASH method recently described by our group to perform a large-scale assessment of the TALEN platform for mutating zebrafish genes (18
Our results show that both homodimeric and heterodimeric TALENs can efficiently modify their targeted loci in zebrafish. Seven of the 10 homodimeric TALEN pairs constructed induced mutations with efficiencies as high as 76%. For eight of the 10 homodimeric TALEN pairs, we constructed obligate heterodimeric versions using the ELD/KKR FokI nuclease domains. We found that these heterodimeric TALEN pairs exhibited either comparable or, in most cases, increased somatic mutation activities (ranging from a two- to seven-fold increase). This finding was somewhat surprising because it differs from what has been observed with the activities of heterodimeric ELD/KKR ZFNs in human cells where these nucleases show somewhat lower activities than their homodimeric counterparts (24
). The heterodimeric TALENs also appear to exhibit less cytotoxicity than their matched homodimeric counterparts as judged by the percentage of normal embryos observed after injection of TALEN-encoded mRNAs. This finding is consistent with the observation that heterodimeric ZFNs possess improved specificities relative to matched homodimeric ZFNs (24
). Our results show that TALENs assembled on the FLASH platform have a high success rate (as measured by their abilities to induce mutations in somatic zebrafish cells) and that active nucleases can efficiently mutagenize their intended targets, a finding consistent with our recent demonstration that FLASH-assembled TALENs have a >88% success rate for targeting endogenous genes in human cells and that active nucleases induce mutations with high efficiencies (18
). In addition, our findings suggest that heterodimeric ELD/KKR TALENs may be more active and less toxic than their homodimeric counterparts. Taken together, we conclude that heterodimeric ELD/KKR TALENs are preferable to homodimeric TALENs for use in zebrafish.
Our data provide a larger scale demonstration that TALEN-induced mutations can be successfully transmitted through the germline. Previous reports with TALENs in zebrafish have shown germline transmission for only two zebrafish genes (20
). In this report, we show that TALEN-induced mutations in eight different endogenous gene targets can be transmitted through the germline using either homodimeric or heterodimeric TALENs. In some cases, we found that all the fish we screened were founders. Our results suggest that founders can typically be identified after screening as few as 5–10 fish injected with TALEN mRNAs if the somatic mutation rate of the TALENs is above 10%. Nevertheless, transmissible mutations may still occur even if the somatic mutation rate falls below 10%, as in the case of elmo1
and as has been shown in a previous report (20
). In this study, more than 70% of the effective homodimeric or heterodimeric TALEN pairs we tested resulted in a somatic mutation rate above 10%.
Interestingly, we found that one of our TALEN pairs exhibits a high degree of specificity for its intended target site. This pair showed greatly reduced activity at a polymorphic target allele that contains two mismatches from the intended sequence. Targeting efficiency for this polymorphic sequence was decreased significantly for both homodimeric and heterodimeric TALENs. This result indicates that TALENs can exhibit strong specificity for their intended binding sequence even when an alternative target bears as few as two differences. This finding parallels that observed for a TALEN pair targeted to the human CCR5
gene, which showed selectivity against an off-target site that differed by only two mismatches from the intended site (29
). Our result also strongly suggests the exciting possibility that it may be possible to design allele-specific TALENs that can selectively mutagenize one allele when two different alleles are present in a single cell. Additional experiments will be needed to test whether this finding can be generalized to other target sites.
The results of our study demonstrate that the TALEN technology is a simple, robust and efficient platform for performing targeted gene disruption in zebrafish. Our findings lead to some practical suggestions for investigators interested in using this technology: (i) Most genes can be successfully targeted by generating TALENs for just one or two sites, (ii) heterodimeric ELD/KKR TALENs are superior to homodimeric TALENs due to their higher activities and lower toxicities and (iii) founders can be identified by screening as few as a handful of fish. The results of this report, together with another recent study showing that TALENs have an essentially limitless targeting range and can be rapidly assembled in high-throughput using the FLASH assembly method (18
), strongly suggest that it may now be feasible to rapidly create very large collections of mutant zebrafish. We also note that all TALENs used in this and our previous study (19
) are built on a particular architecture that has been shown to yield nucleases that can efficiently modify genes in C. elegans
), rats (30
) and human somatic (18
) and pluripotent stem cells (21
). A number of other architectures for building TALENs have been described (20
), and it will be of interest to see whether TALENs built on these other platforms also show similarly high success rates and efficiencies in zebrafish.