Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN)

Abstract

Background: Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells.

Methodology/principal findings: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts.

Conclusions and significance: In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.

Figure 1. The OPEN Zinc Finger Selection Method.

(A) Schematic overview of OPEN selection for a target DNA site. Zinc finger domains are shown as spheres and associated 3 bp subsites as rectangles. Details provided in the text and in Maeder et al., Mol. Cell 2008. (B) Schematic of the bacterial two-hybrid (B2H) system. ZFP = zinc-finger protein. X and Y = arbitrary interacting proteins.

Figure 2. Toxicity and teratogenicity of OPEN and B1H-selected ZFNs in zebrafish embryos.

Percentages of dead, deformed (“monster”), and normal embryos following injection with the amounts of ZFN RNAs indicated are shown. Percentages were calculated from the number of embryos (n) indicated.

Figure 3. Frequencies and sequences of ZFN-induced mutations in somatic zebrafish cells.

For each gene targeted by ZFNs, the wild-type sequence is shown at the top with ZFN binding sites marked. Deletions are indicated by grey highlighted red dashes and insertions by blue highlighted lower case blue letters. The number of times each wild-type mutant allele was isolated is shown in brackets.

Figure 4. Sequences of ZFN-induced mutations transmitted through the germline.

For each target gene, the wild-type sequence is shown at the top with ZFN binding sites marked and the mutated alleles found in founder progeny are shown below the wild-type sequence. Each mutant sequence shown was isolated from progeny of different founders. Deletions are indicated by grey highlighted red dashes and insertions by blue highlighted lower case blue letters.