| Autor: |
Vallecillo-Viejo IC; a Eugene Bell Center for Regenerative Biology and Tissue Engineering , The Marine Biological Laboratory , Woods Hole , MA , USA.; b Department of Medicine , University of Puerto Rico, Medical Sciences Campus , San Juan , Puerto Rico., Liscovitch-Brauer N; c Raymond and Beverly Sackler School of Physics and Astronomy , Tel Aviv University , Tel Aviv , Israel., Montiel-Gonzalez MF; a Eugene Bell Center for Regenerative Biology and Tissue Engineering , The Marine Biological Laboratory , Woods Hole , MA , USA., Eisenberg E; c Raymond and Beverly Sackler School of Physics and Astronomy , Tel Aviv University , Tel Aviv , Israel., Rosenthal JJC; a Eugene Bell Center for Regenerative Biology and Tissue Engineering , The Marine Biological Laboratory , Woods Hole , MA , USA. |
| Abstrakt: |
Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE. |
