Cas9 activates the p53 pathway and selects for p53-inactivating mutations
Authors: Francisca Vazquez
Aaron R Thorner
Daniel Lam
Rameen Beroukhim
Sasha Pantel
Todd R Golub
Mai Abdusamad
Desiree Davison
Veronica Rendo
Naomi Currimjee
Anwesha Nag
Uri Ben-David
Julian Hess
Sangita Pal
John G Doench
Oana M Enache
Journal: Nature Genetics (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Cas9; CRISPR-Cas9; genome editing; p53 pathway; TP53
Affiliations: Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA, USA., Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA., Genetic Perturbation Platform, Broad Institute of Harvard and MIT, Cambridge, MA, USA., Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA, USA., Harvard Medical School, Boston, MA, USA., Howard Hughes Medical Institute, Chevy Chase, MD, USA., Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA., Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA, USA. ., Department of Human Molecular Genetics & Biochemistry, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. .

Abstract:  Cas9 is commonly introduced into cell lines to enable CRISPR-Cas9-mediated genome editing. Here, we studied the genetic and transcriptional consequences of Cas9 expression itself. Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon introduction of Cas9, specifically in wild-type TP53 (TP53-WT) cell lines. This was confirmed at the messenger RNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that introduction of Cas9 can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53-WT and TP53-null (TP53-/-) cell lines. Lastly, Cas9 was less active in TP53-WT than in TP53-mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR-Cas9-mediated genome editing. 

Retracted: No

PubPeer comments:
 Figure 2 was brought to my attention by a reader. * Red boxes: The SNU466 (wildtype) and SNU466 Cas9 panels appear to show an overlap. The images have a different area of view, a different intensity of green dye, and differently marked cells. Could the authors please explain what happened here? ![file] , 
 Thank you for bringing this error to our attention! Indeed, these images were mistakenly cropped from the same image. We looked into it, realized what happened, and submitted an Author Correction to the journal. The immunofluorescence experiments were conducted in three independent experiments, and images were acquired in two different sessions: images from round 1 were acquired one day and images from rounds 2 and 3 were acquired on a second day. During image acquisition, all files were labeled with a numerical ID. The original image files were opened in ImageJ to increase brightness contrast to be able to see the green FITC signal better and to quantify the fluorescent foci. During this process the exact same adjustments were applied to all images coming from the same batch (parental and Cas9-expressing cells were always analyzed together), to enable a fair comparison between conditions. However, we suspect that the mistake happened at the point of selecting which images to use for the actual figure panel. The same image (image_0070_zoverlay.jpg) was mistakenly used for both the parental and Cas9 images, and we think that the color correction was applied to this file twice (thinking it to be a different file that hadn t been processed yet), explaining why the original images in Fig. 2a had different color intensities. The marking of the positive cells was done in Illustrator during figure preparation, to highlight cells with foci, and they were marked differently as they had different intensities already at that point. To correct this error, we first identified the image as file _0070 which originally belonged to the Cas9-expressing SNU466 cells. We went through all of the images and confirmed that they were properly labeled. We then re-analyzed the SNU466 images. We confirmed that this mistake did not affect any of the conclusions of the paper. We re happy to share all of the original raw images, as well as the spreadsheet that was used for foci quantification, upon request. Thanks again for your alertness, which enabled us to correct the error quickly. , 
 I would like to echo Dr. Ben-David in thanking you for noting this so quickly so that we could fix it quickly. We have included all of the original images [here] . ,