An Xiao

Heritable gene targeting in zebrafish using customized TALENs

699 To the Editor: Studies of targeted gene modifications are of great interest in basic research as well as for clinical and agricultural applications1. In the February issue of Nature Biotechnology, two articles reported genomic modifications using transcription activator-like (TAL) effectors2,3. Using fusion proteins, each comprising a TAL effector DNA binding domain and a FokI cleavage domain, Miller et al.2 reported that TAL effector nucleases (TALENs) successfully disrupted target genes in cultured human cells. Zhang et al.3 showed that TAL effectors can be used to regulate endogenous gene transcription. Compared with zinc-finger proteins4,5, TAL effectors permit more predictable and specific binding to target DNA6, and therefore allow researchers to engineer genomes precisely without the need for laborious screening to identify a DNA binding domain with the requisite specificity. TALENs can induce DNA double-stranded breaks (DSBs) in yeast7. Gene targeting using TALENs has also been achieved in nematodes8 and human pluripotent cells9. However, it has not, to our knowledge, yet been demonstrated in a vertebrate organism. Here we report the use of TALENs to disrupt both of the two endogenous zebrafish genes we targeted and show that the mutations are transmitted through the germ line. The complexity of constructing customized, sequence-specific TAL effectors restricts broad application of TALEN to genome engineering. Recently, several strategies for constructing TAL effector repeats using type IIS endonucleases have been reported3,10–14. As an alternative approach, we constructed sequence-specific TAL effector repeats using a method called unit assembly, which involves the isocaudamer restriction enzymes, NheI and SpeI (Supplementary Fig. 1). This method involves four basic single-unit vectors, which recognize the individual nucleotides A, T, C and G (Supplementary Table 1 and Supplementary Sequences). Simple double-restriction-enzyme digestion (NheI + HindIII or SpeI + HindIII), followed by ligation, yields a collection of double-unit elements that each recognize two tandem nucleotides (Supplementary Fig. 1). Having prepared all 16 possible combinations of double units, we used these elements for subsequent construction of TAL effector repeats by serial cycles of digestion and ligation. To construct the TALEN expression vectors, we subcloned the TAL effector repeats into a vector containing the FokI cleavage domain and other necessary components, including the 5′ terminal sequence, the last 0.5 unit encoding the repeat variable di-residue (RVD) NG and the 3′ terminal sequence of pthA from Xanthomonas axonopodis pv. citri (Supplementary Fig. 2 and Supplementary Methods). We selected tnikb (GenBank gene: 556959), which encodes TRAF2 and NCK interacting kinase, as an endogenous gene to test whether customized TALENs can modify the zebrafish (Danio rerio) germ line. The site we chose to target is located at the junction of intron 1 and exon 2, with 15 bp and 16 bp of DNA on the left and the right binding sites, respectively. These are separated by a 15-bp spacer DNA containing a BamHI site (Fig. 1a and Supplementary Fig. 3). To detect mutations, we PCR amplified a 353-bp DNA fragment. Complete digestion with BamHI produced two fragments of 258 bp and 95 bp, as shown in control embryos (Fig. 1). By contrast, there was an apparently intact DNA fragment in embryos injected with mRNA encoding TALENs (Fig. 1) and sequencing results confirmed that indels were induced at the target site (Supplementary Fig. 4). These results confirmed that TALENs could induce DSBs and activate the DNA repair pathway through nonhomologous endjoining in vivo. The NK RVD has been reported to bind to G more specifically than NN does2,15. We constructed a pair of alternative TALENs that binds to the same target site of tnikb by replacing the NN RVD with NK (Supplementary Fig. 3). Survival rates were similar for embryos injected with mRNAs encoding either NNor NKcontaining TALENs (Fig. 1b). However, Heritable gene targeting in zebrafish using customized TALENs

Exdpf Is a Key Regulator of Exocrine Pancreas Development Controlled by Retinoic Acid and ptf1a in Zebrafish

Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21Cip, p27Kip, and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis.

Gfi1.1 regulates hematopoietic lineage differentiation during zebrafish embryogenesis

Growth factor independence 1 (GFI1) is important for maturation of mammalian lymphocytes and neutrophils and maintenance of adult hematopoietic stem cells (HSCs). The role of GFI1 in embryonic hematopoiesis is less well characterized. Through an enhancer trap screen and bioinformatics analysis, we identified a zebrafish homolog of Gfi1 (named gfi1.1) and analyzed its function during embryonic development. Expression of both an endogenous gfi1.1 gene and a GFP reporter gene inserted near its genomic locus was detected in hematopoietic cells of zebrafish embryos. Morpholino (MO) knockdown of gfi1.1 reduced expression of scl, lmo2, c-myb, mpo, rag1, gata1 and hemoglobin alpha embryonic-1 (hbae1), as well as the total amount of embryonic hemoglobin, but increased expression of pu.1 and l-plastin. Under the same conditions, MO injection did not affect the markers involved in vascular and pronephric development. Conversely, overexpression of gfi1.1 via mRNA injection enhanced expression of gata1 but inhibited expression of pu.1. These findings suggest that Gfi1.1 plays a critical role in regulating the balance of embryonic erythroid and myeloid lineage determination, and is also required for the differentiation of lymphocytes and granulocytes during zebrafish embryogenesis.

Identification and expression analysis of mical family genes in zebrafish

Mical (molecule interacting with CasL) represent a conserved family of cytosolic multidomain proteins that has been shown to be associated with a variety of cellular processes, including axon guidance, cell movement, cell-cell junction formation, vesicle trafficking and cancer cell metastasis. However, the expression and function of these genes during embryonic development have not been comprehensively characterized, especially in vertebrate species, although some limited in vivo studies have been carried out in neural and musculature systems of Drosophila and in neural systems of vertebrates. So far, no mical family homologs have been reported in zebrafish, an ideal vertebrate model for the study of developmental processes. Here we report eight homologs of mical family genes in zebrafish and their expression profiles during embryonic development. Consistent with the findings in Drosophila and mammals, most zebrafish mical family genes display expression in neural and musculature systems. In addition, five mical homologs are detected in heart, and one, micall2a, in blood vessels. Our data established an important basis for further functional studies of mical family genes in zebrafish, and suggest a possible role for mical genes in cardiovascular development.

Targeted Mutagenesis in Zebrafish by TALENs.

Zebrafish is a valuable model organism to study vertebrate development, organ regeneration and to generate human disease models. As an important member of the arsenal of genome editing, TALE nucleases (TALENs) have implicated in broad applications in zebrafish reverse genetic studies. In this chapter, we describe the detailed protocols of TALEN-mediated genome manipulations in zebrafish, including targeted gene disruption by indel mutations, deletion of large genomic regions by using two pairs of TALENs, and precise genome modification by homologous recombination (HR).

Progress in zinc finger nuclease engineering for targeted genome modification

Zinc finger nuclease (ZFN) is an artificially engineered hybrid protein that contains a zinc finger protein (ZFP) domain and a Fok I endonuclease cleavage domain. It has recently emerged as a powerful molecular tool for targeted genome modifications. ZFNs recognize and bind to specific DNA sequences to generate a double-strand break (DSB) by its nuclease activity. Based on this finding, various genetic methods, including gene targeting (gene disruption), gene addition, gene correction etc., are being designed to manipulate the genomes of different species at specific loci. One particular advantage of this new technique is its broad applications, which can be employed to generate desirable inheritable mutations both at the organismal level and at the cellular level. Here, we review the recent progress and prospects of ZFN technology. This article focused on the mechanism of how it works, currently available target assessment, ZFP library construction and screening methods, target modification strategies, as well as a collection of specie and genes that have been successfully modified by ZFN. This review will provide a useful reference for researchers who are interested in applying this new technique in their studies.

Generation of Targeted Genomic Deletions Through CRISPR/Cas System in Zebrafish

Using TALEN or CRISPR/Cas system to induce small indels into coding sequences has been implicated in broad applications for reverse genetic studies of many organisms including zebrafish. However, complete deletion of a large gene or noncoding gene(s) or removing a large genomic fragment spanning several genes or other chromosomal elements is preferred in various cases, as well as inducing chromosomal inversions. Here, we describe the detailed protocols for the generation of chromosomal deletion mutations mediated by Cas9 and a pair of gRNAs and the evaluation for the efficiencies in F0 founder fish and of germline transmission.