Light Activated CRISPR System in Zebrafish

Zebrafish is an excellent model for in vivo optogenetic applications because they are transparent at the embryonic and larval stages and develop outside the uterus to provide light access to the whole organism. This optical clarity has been used to directly monitor fluorescent labeled cells and proteins in organisms, so as to gain an in-depth understanding of cellular and subcellular processes in development and disease. In addition to this passive observation, new optogenetic tools can now manipulate biological processes in vivo and directly read out effects.

Figure：CRISPR activation system (a) (Dong, C.,2018)

The role of gene regulatory networks is to determine the genome to be expressed. They affect the development of zebrafish and regulate the expression of thousands of genes in any development process of fish cells. CRISPR activation (CRISPRa) system is a convenient tool for targeted gene activation. It has been developed and combined with lighting based system.

Creative BioMart has been able to make zebrafish a research tool of optogenetic by establishing a light activated CRISPRa system in zebrafish cells.

Basic Principle

We applied the combination of ClB1/CRY switching system and CRISPRa to zebrafish embryonic fibroblasts. The system consists of dcas9 fusion, activation domain and sgRNA. Dcas9 fused with cib1 and the activation domain fused with CRY2.

In the absence of blue light irradiation, there was no interaction between cib1 and CRY2. When ClB1/CRY is exposed to blue light, it will dimerize, guide the RNA binding dcas9 fusion upstream of the transcription initiation site of the desired gene, guide the complex to the target site and activate the desired gene.

The Basic Workflow

When using optogenetic tools, installing low light intensity can minimize possible toxicity. The basic helix ring helix 1 (CIB1) of CRY2 and its binding partner cryptochrome interaction can be induced by blue light in the wavelength range of 390 nm to 480 nm.

Step	Service Description
Plasmid construction	Insert the target fragment into the vector. After the ligation process, competent E. coli cells were used for bacterial transformation. The constructed plasmid sequence was verified by DNA sequencing.
Cell culture and transfection	Plasmid DNA was transferred into target cells using electroporation transfection or nuclear transfection. The cells were treated with dark light
RT and qPCR test	Check the mRNA expression of the gene of interest

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Reference

Dong, C., Fontana, J., Patel, A. et al. Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria. Nat Commun 9, 2489 (2018).

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