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.

Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs)

“…ELD/ KKR are derived from the Fok I nuclease domain and are reported to have higher specificity (16) and less toxicity (17) than Fok I homodimers. Although it was reported previously that these modified Fok I domains exhibited decreased cleavage efficiency in human cells, recent studies indicated that ELD/KKR showed higher activities than wild-type Fok I in zebrafish embryos, and that TALENs containing EL/KK or ELK/KKR induced a similar rate of deformed and dead embryos in zebrafish (17). The ZFN pairs we tested contained EL and KK, respectively.…”

“…S1). ELD and KKR have less off-target effects and higher efficiency because of compulsory heterodimer formation (16,17). Thus, through dimerization of ELD and KKR, a pair of TALENs that binds to upstream and downstream EBEs of a given site is expected to cleave the DNA in the spacer region between the two EBEs (Fig.…”

A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish

“…More recently, specific genomic DSB has also been successfully generated by using RNA-guided Cas9 nuclease, providing an alternative approach for genome editing [4,5]. To date, genome editing mediated by these engineered nucleases has been successfully implemented in cells and in a number of organisms including Xenopus tropicalis and zebrafish [6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”

Reverse genetics tools in zebrafish: A forward dive into endocrinology

“…TALENs use the same functional domains, namely the restriction enzyme domain and the DNA binding domain. In the case of TALENs, the DNA binding domain is derived from plant pathogenic bacteria with TALE modules recognizing specific bases ( Figure 2B) [4]. TALENs are far more flexible and much simpler to generate than ZFNs because each TALE module of the DNA recognition domain of TALENs consists of a cipher for one particular DNA base and can be easily assembled one by one.…”