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Controlled gene editing has the potential to transform disease treatment, crop climate tolerance, and our ability to investigate genetics. The CRISPR-Cas9 gene editing system has proven to be a key advance due to the ease and precision of making specific genetic changes. Now, two independent teams demonstrate that CRISPR-Cas9 can be activated only in a subset of cells in culture and in zebrafish embryos at specific times, controlling the system with light more strongly than previous attempts (ACS Cent. Sci. 2020, DOI: 10.1021 / acscentsci.9b01093; Angew Chem In t. Ed. 2020, DOI: 10.1002 / anie.201914575). Unlike previous efforts, the teams method targets a part of the CRISPR-Cas9 system known as the guide RNA.
Light offers many advantages as a switch to control gene editing in cell culture or tissue: it is minimally invasive, it works quickly, and, unlike biochemical controls, it does not need additional chemical agents added to cells. The ability to manipulate where and when genes are edited could help researchers attack subsets of cells within an organ, or only manipulate a gene at certain times during embryonic development. The researchers have already developed a variety of light-activated CRISPR-Cas9 systems, all of which involve light-controlled Cas9 proteins. But it is difficult to make additional updates that would improve the selectivity or efficiency of gene editing for light-controlled proteins. Therefore, it would be better to add the light control elsewhere to allow researchers to more freely design Cas9, says Alexander Deiters of the University of Pittsburgh, who led one of the studies.
The teams then focused on a part of the system called guide RNA, which is designed to bind to the DNA sequence it is meant to cut, creating a DNA-RNA complex that the Cas9 enzyme recognizes and cuts. In the new studies, teams led by Deiters and Molly M. Stevens of the Karolinska Institute modified the guide RNA molecules, bringing light-sensitive molecules that “caged” the RNA to prevent DNA pairing. When exposed to the correct wavelength of light, the caged molecules separate and the RNA can bind to the target DNA, initiating the gene-editing process.
“Essentially they install this pretty photoclinable button on the RNA bases involved in the Watson-Crick pairing,” which is necessary for Cas9 to cut DNA, said chemical biologist Amit Choudhary of the Broad Institute, who was not involved in any of the studies. , speaking of The Stevens Group Work. “In the absence of light, pairing does not occur and there is very little background activity.”
The researchers treated cultured human cells with the photoenaged RNAs, and found no signs of DNA editing in the absence of light. When illuminated, the activated RNA restored 70% of the gene editing activity observed with control RNA molecules that lacked the photoactive attachments in the Stevens group experiments. Both groups tested their systems on zebrafish embryos and found that they could introduce mutations into one eye and not the other by illuminating it. Similar results from two independent studies such as this are “particularly reassuring” that the method will be reproducible, Lapatrada Taemaitree and Tom Brown of the University of Oxford wrote in one perspective (ACS Cent. Sci. 2020, DOI: 10.1021 / acscentsci.0c00350). “In principle, multi-color targeting systems could be developed to allow different genes to be turned off simultaneously or at different times.”
