Randall T. Peterson

Efficient genome editing in zebrafish using a CRISPR-Cas system

In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator–like effector nucleases.In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases.

In vivo drug discovery in the zebrafish

The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which large, phenotype-based screens can be performed. Because of these attributes, the zebrafish might also provide opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

Targeted gene disruption in somatic zebrafish cells using engineered TALENs.

To the Editor: Miller et al. recently described a TALE nuclease architecture for performing efficient genome editing1. The authors demonstrated that TALE nucleases, composed of an engineered array of TALE repeats fused to the non-specific FokI cleavage domain, could be used to introduce targeted double-stranded breaks (DSBs) in human cells with high efficiency. Repair of these DSBs by normal DNA repair mechanisms such as non-homologous end-joining (NHEJ) or homologous recombination (HR) enables introduction of alterations at or near the site of the break. A single 34 amino acid TALE repeat binds to one bp of DNA and repeats that bind each of the four DNA bases have been described2, 3. These modules can be assembled into arrays capable of binding extended DNA sequences. TALE nucleases may have advantages over engineered zinc finger nucleases (ZFNs) due to the relative ease with which they can be designed and their potential ability to be targeted to a wide range of sequences (with target sites reported to be as frequent as 1 in 35 bps of random DNA sequence4). We sought to determine whether the TALE nuclease framework described by Miller et al. could also be used to efficiently modify endogenous genes in zebrafish. Previous studies have shown that error-prone repair of ZFN-induced DSBs by NHEJ can result in the efficient introduction of small insertions or deletions (indels) at cleavage sites in endogenous zebrafish genes5–7. These indels frequently result in frameshift knockout mutations that can be passed through the germline to create mutant fish5–9. ZFN technology has enabled reverse genetics studies to be performed in zebrafish, a capability that did not previously exist. However, engineering ZFNs can be challenging due to the need to account for context-dependent effects among individual fingers in an array. In addition, although many zebrafish genes can be targeted with ZFNs made by publicly available methods that account for context-dependence7, 10, it can in some instances be difficult to target within some genes in zebrafish due to the currently limited targeting range of publicly available ZFN engineering platforms. Thus, if TALE nucleases could be used to introduce targeted mutations in zebrafish, this platform would provide an important additional capability for this model organism. To test the ability of TALE nucleases to function in zebrafish, we targeted DNA sequences in two endogenous zebrafish genes gria3a and hey2 (Figure 1). To avoid confounding effects that might affect binding and cleavage of DNA sites by TALE nucleases (e.g.--chromatin structure or DNA methylation), we chose to target sequences that we had efficiently altered previously in zebrafish using engineered ZFNs (Supplementary Figures 1 and 2). Using an iterative assembly approach (Supplementary Methods), we constructed four TALE nuclease monomers to partially overlapping sites in the gria3a gene and two TALE nuclease monomers to a site in the hey2 gene (Figure 1 and Supplementary Figure 3). These six TALE nuclease monomers all harbor the wild-type FokI cleavage domain (Supplementary Figures 4 and 5) and can be paired in combinations to make three TALE nuclease dimers to the gria3a gene and one TALE nuclease dimer to the hey2 gene (Figure 1). We injected RNAs encoding the various TALE nuclease pairs into one-cell stage zebrafish embryos and determined the frequency of NHEJ-mediated mutagenesis at the target site by sequence analysis of alleles from pooled injected embryos (Supplementary Methods, Supplementary Figs. 6–10 and Supplementary Table 1). As shown in Figure 1, we found that all four pairs of TALE nucleases induced targeted indels with high mutation frequencies ranging from 11 to 33%. These frequencies are comparable to what we obtained with ZFNs targeted to DNA sequences in the same vicinity of the gene (Supplementary Figure 1); however, we note that TALE nucleases harbor wild-type FokI domains whereas the ZFNs harbor obligate heterodimeric FokI domains11. Although small indels were typically observed with the TALE nucleases, some large deletions (up to 303 basepairs) were also found (Figure 1). Figure 1 Target sequences, frequencies of mutations, and sequences of mutations induced by TALE nucleases in embryonic zebrafish cells To assess the toxicity of our engineered TALE nucleases, we scored the percentages of dead and deformed embryos that resulted from mRNA microinjections (Supplementary Figure 11). Although we cannot directly compare these results with the microinjections of ZFNs due to the differences between the FokI endonuclease domains used (EL/KK heterodimeric FokI11 for ZFNs versus wild-type FokI for the TALE nucleases) and the specific sequences targeted, the toxicity we observed with injection of 600 pg of TALE nuclease mRNAs (ranging between 40–80%) appears similar to that observed with 400–500 pg of mRNAs encoding ZFNs targeted to sequences in the same vicinity and to other genes (Supplementary Figure 12 and Reference 7). An important future experiment will be to demonstrate germline transmission of TALE nuclease-induced mutations. Given that the frequencies of mutation and the extent of toxicities we observe are similar to what we have seen with ZFNs, we expect that TALE nuclease-induced mutations should be efficiently passed through the germline to progeny and we are currently conducting experiments to test this prediction. Successful germline transmission of these mutations will be critical for using TALE nucleases to perform reverse genetics in zebrafish. Progeny fish bearing TALE nuclease-induced mutations, unlike founder F0 fish, will not be mosaic (i.e.--these fish will have uniform mutation of all cells in the organism); such mutant fish will enable determination of whether both mono-allelic and bi-allelic alterations of a gene are possible and will provide a more straightforward background on which to perform analysis of off-target effects. In summary, we show that the TALE nuclease framework described by Miller et al. can be used to efficiently introduce targeted indel mutations in endogenous zebrafish genes at the somatic cell level. Although in this study we chose two genomic loci that have been successfully targeted with ZFNs before, all six TALE nuclease monomers we constructed showed high mutagenesis activities when tested in various pairwise combinations, suggesting that the TALE nuclease framework is also highly robust and effective in zebrafish. As is the case with ZFNs, the complete genome-wide spectrum of off-target mutations introduced by TALE nucleases remains unknown. However, expression of the TALE nucleases we made in zebrafish does not show toxicity substantially different from that observed with expression of ZFNs, suggesting that the magnitude of off-target effects may be comparable with the two types of nucleases. In principle, off-target mutations made by TALE nucleases can be removed by out-crossing the founder assuming that they are not tightly linked to the intended mutation. In addition, mutant phenotypes could also be confirmed by generation of a second mutant allele using nucleases targeted to a different site. For mutagenesis of genes in zebrafish (and other model organisms such as C. elegans12), TALE nucleases may offer potential advantages over ZFNs because they can be easily and quickly assembled in a modular fashion and they can potentially target a greater range of DNA sequences. Thus, we expect that the ability to utilize both ZFNs and TALE nucleases should enable any researcher to rapidly and easily create targeted mutations in their endogenous zebrafish gene of interest.

Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation

Behavioral Profiling The complexity of the brain makes it difficult to predict how a drug will affect behavior without direct testing in live animals. Rihel et al. (p. 348) developed a high-throughput assay to assess the effects of thousands of drugs on sleep/wake behaviors of zebrafish larvae. The data set reveals a broad conservation of zebrafish and mammalian sleep/wake pharmacology and identifies pathways that regulate sleep. Moreover, the biological targets of poorly characterized small molecules can be predicted by matching their behavioral profiles to those of well-known drugs. Thus, behavioral profiling in zebrafish offers a cost-effective way to characterize neuroactive drugs and to predict biological targets of novel compounds. The effects of most neuroactive drugs are conserved and can be detected by behavioral screening. A major obstacle for the discovery of psychoactive drugs is the inability to predict how small molecules will alter complex behaviors. We report the development and application of a high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found that the multidimensional nature of observed phenotypes enabled the hierarchical clustering of molecules according to shared behaviors. Behavioral profiling revealed conserved functions of psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds. In addition, behavioral profiling implicated new factors such as ether-a-go-go–related gene (ERG) potassium channels and immunomodulators in the control of rest and locomotor activity. These results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors.

BMP type I receptor inhibition reduces heterotopic ossification

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2). Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.

Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation

Conventional drug discovery approaches require a priori selection of an appropriate molecular target, but it is often not obvious which biological pathways must be targeted to reverse a disease phenotype. Phenotype-based screens offer the potential to identify pathways and potential therapies that influence disease processes. The zebrafish mutation gridlock (grl, affecting the gene hey2) disrupts aortic blood flow in a region and physiological manner akin to aortic coarctation in humans. Here we use a whole-organism, phenotype-based, small-molecule screen to discover a class of compounds that suppress the coarctation phenotype and permit survival to adulthood. These compounds function during the specification and migration of angioblasts. They act to upregulate expression of vascular endothelial growth factor (VEGF), and the activation of the VEGF pathway is sufficient to suppress the gridlock phenotype. Thus, organism-based screens allow the discovery of small molecules that ameliorate complex dysmorphic syndromes even without targeting the affected gene directly.

Drugs That Induce Repolarization Abnormalities Cause Bradycardia in Zebrafish

Background—Drug-induced QT prolongation and torsades de pointes remain significant and often unpredictable clinical problems. Current in vitro preclinical assays are limited by biological simplicity, and in vivo models suffer from expense and low throughput. Methods and Results—During a screen for the effects of 100 small molecules on the heart rate of the zebrafish, Danio rerio, we found that drugs that cause QT prolongation in humans consistently caused bradycardia and AV block in the zebrafish. Of 23 such drugs tested, 18 were positive in this initial screen. Poor absorption explained 4 of 5 false-negative results, as demonstrated by microinjection. Overall, 22 of 23 compounds that cause repolarization abnormalities were positive in this assay. Antisense “knockdown” of the zebrafish KCNH2 ortholog yielded bradycardia in a dose dependent manner confirming the effects of reduction of repolarizing potassium current in this model. Classical drug-drug interactions between erythromycin and cisapride, as well as cimetidine and terfenadine, were also reproduced. Conclusion—This simple high-throughput assay is a promising addition to the repertoire of preclinical tests for drug-induced repolarization abnormalities. The genetic tractability of the zebrafish will allow the exploration of heritable modifiers of such drug effects.

FKBP12-Rapamycin-associated Protein (FRAP) Autophosphorylates at Serine 2481 under Translationally Repressive Conditions

The FKBP12-rapamycin associated protein (FRAP, also RAFT, mTOR) belongs to a family of phosphatidylinositol kinase-related kinases. These kinases mediate cellular responses to stresses such as DNA damage and nutrient deprivation in a variety of eukaryotes from yeast to humans. FRAP regulates G1cell cycle progression and translation initiation in part by controlling the phosphorylation states of a number of translational and cell cycle regulators. Although FRAP is known to be phosphorylatedin vivo and to phosphorylate several proteins (including itself) in vitro, FRAPs phosphorylation sites and substrate specificity are unknown. We report here the identification of a FRAP autophosphorylation site. This site, Ser-2481, is located in a hydrophobic region near the conserved carboxyl-terminal FRAP tail. We demonstrate that the COOH-terminal tail is required for FRAP kinase activity and for signaling to the translational regulator p70s6k (ribosomal subunit S6 kinase). Phosphorylation of wild-type but not kinase-inactive FRAP occurs at Ser-2481 in vivo, suggesting that Ser-2481 phosphorylation is a marker of FRAP autokinase activity in cells. FRAP autophosphorylation is blocked completely by wortmannin treatment but not by rapamycin treatment, amino acid deprivation, or serum withdrawal, treatments that lead to acute dephosphorylation of eIF4E-binding protein (4E-BP1) and p70s6k. Ser-2481 phosphorylation increases slightly upon c-Akt/PKB activation and dramatically upon calyculin A treatment of T-cells. These results suggest that FRAP-responsive dephosphorylation of 4E-BP1 and p70s6k occurs through a mechanism other than inhibition of intrinsic FRAP kinase activity.