PgmNr Z6032: Opto-CRISPR : a new tool for genome editing at the single cell level.

Authors:
B. Ducos; W. Zhang; D. Bensimon


Institutes
LPS-ENS, CNRS, Paris, FR.


Abstract:

The CRISPR-Cas9 system has recently emerged as an essential tool for genome editing, acting like genetic scissors to precisely abrogate or restore gene function, or transcribe specific targets. An important issue in deciphering gene regulation in time and space is to develop strategies for conditional gene expression. Usually this approach is based on transgenic lines where the expression of the genes of interest is under the control of tissue-specific promoters that can be switched on or off using drugs or heat. While much qualitative information on genetic networks have been obtained by that method, the lack of precise control of the concentration, location and timing of the gene products impair a quantitative analysis of the physiological response to such perturbations.

To overcome this gaps our lab has developed an optical method combining the use of  conditional protein release via a protein-ERT fusion and a caged ligand, namely caged-cyclofen. Upon illumination with UV light (at 370nm) or with a two-photon laser beam, active cyclofen is released in the illuminated cell(s). It binds to the hormone binding domain of a truncated estrogen receptor (ERT) fused to the protein of interest which it releases from the complex it forms with cytoplasmic chaperones. If the protein of interest is a Gal4 transcription factor, it then diffuses to the nucleus, binds to its UAS promoter and turns on the gene of interest (in particular a Cas9 gene) and a fluorescent protein (CFP) reporter expressed as a bi-cistronic gene. This method allows us to specifically express Cas9 in live zebrafish in a controlled manner at given time and spatial location and to visualize the photoactivated cells.

We shall present results where we co-injected UAS:Cas9-T2A-CFP-Ubi:EosFP and gRNA for EosFP gene at one cell stage. Caged-cyclofen was UV activated at 256 cells and the  embryos imaged at 24 hpf. CFP (blue) fluorescence was observed in the photoactivated cells and was negatively correlated with EosFP green fluorescence suggesting that in these cells Cas9 was active as indeed confirmed by T7 endonuclease analysis.

We have also developed a direct photo-switchable Cas9 by fusing its gene with ERT. In this context while Cas9 is continuously expressed, it is sequestered by cytoplasmic chaperones and quickly released at the single cell level upon cyclofen uncaging.

These new tools are currently developed as zebrafish transgenic lines offering the zebrafish community a powerful tool to investigate gene functions and networks (by crossing with relevant gRNA lines or by injections of appropriate gRNAs).



ZFIN Genetics Index
1. Cas9
2. Gal4FF
3. ERT
4. CFP
5. EosFP
6. t2a