Chromosomes lighting up
A rotating 3D model of a cell with the Casilio labeling of telomeres generated from a stack of confocal images. The second half of the model shows spots identified by the image analysis software Imaris, which converted the signal into 3D coordinates for further computational analysis.
A new gene regulation and labeling platform dubbed “Casilio” can simultaneously execute distinct functions at multiple areas of the genome at the same time, expanding researchers’ ability to study gene function and chromosome structure, Jackson Laboratory (JAX) researchers report.
The widely used CRISPR-Cas9 gene-editing technology has enabled researchers to alter genome sequence or gene expression with great precision, says JAX Assistant Professor Albert Cheng, Ph.D., first and co-corresponding author of a paper published in Cell Research. “However, CRISPR currently can carry out only one function in the cell at a time. But most cell functions are more complex than that—being regulated by multiple genes.”
Working with JAX Distinguished Visiting Professor Haoyi Wang, Ph.D., and other colleagues, Cheng was looking for a way to regulate multiple genes in the mouse and other cell and animal models. The new system, called Casilio, combines an altered version of CRISPR (CRISPR-dCas9) with the Pumilio RNA-binding protein system to enable much broader gene manipulation power.
“If CRISPR’s action could be compared to a simple flute melody, Casilio’s would be a Beethoven symphony,” Cheng notes. “Casilio ‘orchestrates’ variations throughout the genome, simultaneously and in high fidelity. That means one-step modeling of complex gene expression profiles.”
CRISPR-Cas9 cuts DNA at a precise location, guided there by a sequence-specific RNA. dCas9 has been modified so that it no longer cuts DNA, but it can still be guided to specific sequences and will bind to them. dCas9 can be fused to protein tags or the effector domains of transcription factors (proteins that bind DNA in promoter regions and simultaneously bind other regulatory proteins via effector domains, which can activate or repress transcription and thereby affect gene expression) to develop customized DNA binding proteins.
For example, it can be fused to domains that activate or repress gene expression, allowing researchers to increase or decrease gene expression at will without changing the actual sequence. (Many diseases involve variations in gene expression rather than genetic sequence.) It can also be fused to fluorescent proteins to label specific chromosomal regions in live cells for study.
Previously only one type of Cas9-effector function has been possible, and it’s hard to recruit multiple copies of effectors or protein tags, though they are sometimes necessary for sufficient activity.
The Pumilio RNA-binding protein system allows the design of a simple nucleotide code that recognizes an eight-nucleotide RNA sequence, called a PUF domain. Adding Pumilio binding sites to the guide RNA in the CRISPR system allows tethering of PUF domains fused with effector domains or protein tags.
With this feature, Casilio can now conduct different functions at different genomic sites--e.g., upregulating one set of genes while repressing another set. Using single guide RNA molecules with multiple numbers of Pumilio binding sites, Casilio can recruit several effector molecules to target sites. This advantage enables more efficient gene modulation and labeling of chromosomal loci in live cells.
Cheng plans to launch a web presence for Casilio at http://casil.io/ to collect citations and news from his lab and others using the Casilio system.
He also hopes to expand the number of Casilio modules available to researchers. “Future modules should be orthogonal in that every module has a unique PUF-RNA pairing, he says. “I would also like to have a module database, like an App Store, where researchers can ‘shop’ for Casilio modules – e.g., modules for adding acetylation, removing methylation, inducing double-strand breaks, etc., just plug-and-play, mix and match the different modules to do something fun in the cell. I think this expandability is a very important feature of Casilio.”
Cheng et al.: Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cell Research advance online publication, Friday, January 15, 2016, doi: 10.1038/cr.2016.3.