Lee, Han

Abstract The vertebrate stress response (SR) is mediated by the hypothalamic-pituitary-adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. The glucocorticoid receptor (nr3c1) was necessary to maintain basal locomotor activity in light and darkness. The HPA axis was required to adapt to light more efficiently but became dispensable when longer illumination was provided. Light adaptation was more efficient in dimmer light and did not require the mineralocorticoid receptor (nr3c2). Our findings show that the HPA axis contributes to the SR at various stages, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPA axis activity.

Nearly 90% of human pathogenic mutations are caused by small genetic variations, and methods to correct these errors efficiently are critically important. One way to make small DNA changes is providing a single-stranded oligo deoxynucleotide (ssODN) containing an alteration coupled with a targeted double-strand break (DSB) at the target locus in the genome. Coupling an ssODN donor with a CRISPR-Cas9-mediated DSB is one of the most streamlined approaches to introduce small changes. However, in many systems, this approach is inefficient and introduces imprecise repair at the genetic junctions. We herein report a technology that uses spatiotemporal localization of an ssODN with CRISPR-Cas9 to improve gene alteration. We show that by fusing an ssODN template to the trans-activating RNA (tracrRNA), we recover precise genetic alterations, with increased integration and precision in vitro and in vivo . Finally, we show that this technology can be used to enhance gene conversion with other gene editing tools such as transcription activator like effector nucleases.

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic‐pituitary‐adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic‐pituitary‐adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic‐pituitary‐interrenal (HPI) axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived eight frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 ofmc2r(adrenocorticotropic hormone receptor), five in exon 2 or 5 ofnr3c1(glucocorticoid receptor [GR]) and two in exon 2 ofnr3c2(mineralocorticoid receptor [MR]). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning HPI axis via the action ofmc2rand the canonical GR encoded bynr3c1gene, but not MR (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic‐pituitary‐adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.

Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (C t ) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.