Never has a discovery taken the world of biotechnology by storm like the photoactivatable CRISPR-Cas9 systems. This tool has been around for a while and has been used to control gene expression. But there was no way to turn the transcription on and off. Now there is and it’s simpler than we thought!
Teams led by Moritoshi Sato at the University of Tokyo and Charles Gersbach at Duke University have independently reported the development of CRISPR-Cas9-based transcriptional activation systems that could be controlled with light.
Complex gene networks are essential for diverse biological phenomena such as cellular programming, metabolism, homeostasis, memory formation, and circadian rhythm. To understand these gene functions, methods that allow endogenous gene expression regulation at will are essential. Molecular tools like CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) system in bacteria have been of great help so far. They are a class of programmable genome targeting technology used to enable endogenous gene expression.
The Cas9 part of this system is a RNA-directed DNA-binding protein guided by a single guide RNA. By inactivating its nuclease activity, coupling the protein to other effector domains, and choosing an appropriate guide sequence, researchers direct these activities wherever they wish in the genome.
This system also consists of two fusion proteins: one couples an inactivated Cas9 protein to a protein called CIB1 and the other couples a transcriptional activation domain to cryptochrome 2 (CRY2). When the cells expressing the above two proteins and a guide RNA are illuminated with blue light, the two proteins pair up and activate transcription by binding to the transcription activation domain of the DNA.
Both Sato and Gersbach also demonstrated that this transcription can be controlled by photomasking the illumination in specific regions of the culture plate. “This method allows targeted, [user]-defined gene activation by light and could contribute to many biomedical applications, such as exploring gene functions and utilizing them to control cellular functions in vitro and in vivo,” Sato said.
The original paper can be accessed here.