By now, most within the biomedical fields have likely at least heard mention of the gene-editing tool known as CRISPR/Cas9. Since 2013, this technique has created a veritable revolution within the disciplines of molecular biology and genetics and is already showing tremendous promise for treating such diseases a cystic fibrosis and sickle cycle, as well as being able to generate new animal models that better mimic human disease.
The power of the CRISPR/Cas9 system lies not only in its powerful editing capabilities, but also its speed, simplicity, and compatibility for use within most organisms. However, there is one main drawback for which scientists at the University of Pittsburgh and University of North Carolina believe they may have just found a solution—conditional control, or making the system tunable, using light.
The findings from this study were published recently in the Journal of the American Chemical Society through an article entitled “Optical Control of CRISPR/Cas9 Gene Editing.”
Conventionally, this gene-editing technique uses the bacterially derived Cas9 protein along with a synthetic guide RNA to introduce a double-strand break at the desired location within the genome. This allows for the excision of a stretch of DNA or some alteration of gene function through the introduction of mutations within that region.
The standard process is an all or nothing approach, where investigators select for the genotype or phenotype of interest after treating cells with the CRISPR/Cas9 assay. In the current study however, the researchers created a functional inactive Cas9 protein that can be turned on in the presence of a specific wavelength of light.
“In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid,” stated the scientists. “Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function.”
The research team noted that the improved control over the time and location at which a gene can be manipulated may offer a solution to off-target effects, as well as potentially enable genetic studies with unparalleled resolution.
“This method may allow people to engineer genes in cells or animals with better spatial and temporal control than ever before,” explained Alexander Deiters, Ph.D., professor of chemistry at the University of Pittsburgh and sensor author on the current study. “Previously, if you wanted to knock out a gene, you had limited control over where and when it would happen. Engineering a light switch into Cas9 provides a more precise editing tool. You can say, 'In this cell, at this time point, is where I want to modify the genome.'”