Zuoyan Zhu

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

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.

TALEN-mediated precise genome modification by homologous recombination in zebrafish.

We report gene targeting via homologous recombination in zebrafish. We co-injected fertilized eggs with transcription activator–like effector nuclease mRNAs and a donor vector with long homologous arms targeting the tyrosine hydroxylase (th) locus, and we observed effective gene modification that was transmitted through the germ line. We also successfully targeted two additional genes. Homologous recombination in zebrafish with a dsDNA donor expands the utility of this model organism.

CasOT: a genome-wide Cas9/gRNA off-target searching tool

The CRISPR/Cas or Cas9/guide RNA system is a newly developed, easily engineered and highly effective tool for gene targeting; it has considerable off-target effects in cultured human cells and in several organisms. However, the Cas9/guide RNA target site is too short for existing alignment tools to exhaustively and effectively identify potential off-target sites. CasOT is a local tool designed to find potential off-target sites in any given genome or user-provided sequence, with user-specified types of protospacer adjacent motif, and number of mismatches allowed in the seed and non-seed regions. AVAILABILITY http://eendb.zfgenetics.org/casot/ CONTACT: zfgenetics@gmail.com or bzhang@pku.edu.cn Supplementary Information: Supplementary data are available at Bioinformatics online.

Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinsons disease.

Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions

Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F1 offspring of the infected fish. From F1 fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes. By inbreeding fish carrying proviral integrations in 25 different genes, we were able to demonstrate that in ≈50% of the gene “hits,” the mRNA transcript levels were reduced by ≥70%, with the highest probability for mutation occurring if the integration was in an exon or first intron. Based on these data, the mutagenic frequency for the retrovirus is nearly one in five integrations. In addition, a strong mutagenic effect is seen when murine leukemia virus integrates specifically in the first intron of genes but not in other introns. Three of 19 gene inactivation events had embryonic defects. Using the strategy we outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F1 fish. This is a 20- to 30-fold increase in efficiency when compared with the current resequencing approach [targeting induced local lesions in genomes (TILLING)] used in zebrafish for identifying mutations in genes. Combining this increase in efficiency with cryopreservation of sperm samples from the F1 fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene.

Screening for genes essential for mouse embryonic stem cell self-renewal using a subtractive RNA interference library.

The pluripotency of mouse embryonic stem (ES) cells is maintained by self‐renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker Oct‐4 were monitored 48 hours later by real time RT‐PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated Oct‐4 expression to less than 50% of the normal level and 7 of them upregulated Oct‐4 expression to more than 150% of the normal level. To investigate their long‐term effect on self‐renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418‐resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including Zfp42/Rex‐1, which was chosen for further functional analysis. RNAi knockdown of Zfp42 induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of Zfp42 also caused ES cells to lose the capacity of self‐renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self‐renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.

EENdb: a database and knowledge base of ZFNs and TALENs for endonuclease engineering

We report here the construction of engineered endonuclease database (EENdb) (http://eendb.zfgenetics.org/), a searchable database and knowledge base for customizable engineered endonucleases (EENs), including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). EENs are artificial nucleases designed to target and cleave specific DNA sequences. EENs have been shown to be a very useful genetic tool for targeted genome modification and have shown great potentials in the applications in basic research, clinical therapies and agricultural utilities, and they are specifically essential for reverse genetics research in species where no other gene targeting techniques are available. EENdb contains over 700 records of all the reported ZFNs and TALENs and related information, such as their target sequences, the peptide components [zinc finger protein-/transcription activator-like effector (TALE)-binding domains, FokI variants and linker peptide/framework], the efficiency and specificity of their activities. The database also lists EEN engineering tools and resources as well as information about forms and types of EENs, EEN screening and construction methods, detection methods for targeting efficiency and many other utilities. The aim of EENdb is to represent a central hub for EEN information and an integrated solution for EEN engineering. These studies may help to extract in-depth properties and common rules regarding ZFN or TALEN efficiency through comparison of the known ZFNs or TALENs.

Vertebrate MAX-1 is required for vascular patterning in zebrafish

During embryogenesis, stereotypic vascular patterning requires guidance cues from neighboring tissues. However, key molecules involved in this process still remain largely elusive. Here, we report molecular cloning, expression, and functional studies of zebrafish max-1, a homolog of Caenorhabditis elegans max-1 that has been implicated in motor neuron axon guidance. During early embryonic development, zebrafish max-1 is specifically expressed in subsets of neuronal tissues, epithelial cells, and developing somites through which vascular endothelial cells migrate from large ventral axial vessels to form stereotypic intersegmental blood vessels (ISV). Blocking zebrafish max-1 mRNA splicing by morpholino injection led to aberrant ISV patterning, which could be rescued by injection of either C. elegans or zebrafish max-1 mRNA. Analysis of motor neurons in the same region showed normal neuronal axon pathfinding. Further studies suggested that the ISV defect caused by max-1 knockdown could be partially rescued by overexpression of ephrinb3 and that max-1 was involved in mediating membrane localization of ephrin proteins, which have been shown to provide guidance cues for endothelial cell migration. Our findings therefore suggest that max-1, acting upstream of the ephrin pathway, is critically required in vascular patterning in vertebrate species.

Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio

Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio