1.
Spatiotemporal control of zebrafish (Danio rerio) gene expression using a light-activated CRISPR activation system.
Abstract:
CRISPR activation (CRISPRa) system is the convenient tool for targeted-gene activation, it has been developed and combined with a lighting-based system that can control transcription initiation spatially and temporally by utilizing photoreceptor derived from plant Arabidopsis thaliana. A blue light photoreceptor the Cryptochrome 2 (CRY2), and its binding partner CIB1 will dimerize by exposure to the blue light and it has been applied to human cells. However, the application of a combination of these two systems to zebrafish cell is still not explored. We performed zebrafish gene activation using p65 and VP64 activators in the zebrafish cells (ZF4). Our study demonstrated that we have successfully controlled the transcription level of ASCL1a, BCL6a, and HSP70 genes using blue light-activated CRISPR activation system. The result showed that using this system, mRNA level expression of ASCL1a, BCL6a, and HSP70 genes increased after irradiated under blue light for several hours and significantly different to those which treated in the dark.
2.
New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research using Zebrafish.
Abstract:
Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope, versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where CRISPR/Cas9 has been applied very extensively for many different purposes and with great success. In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity in a preclinical setting. Here we describe multiple advances in diverse species and systems that can improve genome editing in zebrafish. To achieve temporal and spatial precision of genome editing, many new technologies can be applied in zebrafish such as artificial transcription factors, drug-inducible or optogenetically-driven expression of Cas9, or chemically-inducible activation of Cas9. Moreover, chemically- or optogenetically-inducible reconstitution of dead Cas9 (catalytically inactive, dCas9) can enable spatiotemporal control of gene regulation. In addition to controlling where and when genome editing occurs, using oligonucleotides allows for the introduction (knock-in) of precise modifications of the genome. We review recent trends to improve the precision and efficiency of oligo-based point mutation knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species. We discuss the first examples and prospects of approaches using sgRNAs as specific and effective mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for high-throughput genetic screens.
