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Cas9 activates the p53 pathway and selects for p53-inactivating mutations

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An Author Correction to this article was published on 25 June 2020

This article has been updated

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.

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Fig. 1: Cas9 introduction can activate the p53 pathway.
Fig. 2: Cas9 introduction is associated with elevated DNA damage.
Fig. 3: Cas9 introduction selects for inactivating TP53 mutations.
Fig. 4: Expansion of inactivating TP53 mutations is accelerated by Cas9 in a cell competition assay.
Fig. 5: Cas9-induced p53 activation can functionally affect genetic and chemical perturbation assays.

Data availability

All data sets are available within the article, its Supplementary Information, or from the corresponding authors upon request. DNA sequencing data were deposited to SRA (https://www.ncbi.nlm.nih.gov/sra) with BioProject accession number PRJNA545458. Gene expression data were uploaded to the following URL: https://clue.io/data/XPR_BASE#CAS9_BASELINE. Source Data of all immunostaining blots (in Fig. 1 and Extended Data Fig. 2) are available in the online version of this paper. Raw microscopy data are available at https://figshare.com/projects/Cas9_activates_the_p53_pathway_and_selects_for_p53-inactivating_mutations/81080.

Code availability

All of the code used to generate and/or analyze the data is publicly available.

Change history

  • 25 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank A. Subramanian, D. Root, T. Natoli, C. Kadoch, S. Cassel and C. Collings for helpful discussions; X. Lu, A. Giacomelli, K. Labella, K. Sanson and W. Hahn for contributing reagents; M. Ducar and S. Drinan for assistance with the OncoPanel assay; and F. Piccioni for assistance with Cas9 cell line generation. This work was supported by the NIH (R01 CA18828, CA215489 and CA219943 to R.B.), the Gray Matters Brain Cancer Foundation (R.B.), Pediatric Brain Tumor Foundation (R.B.), HHMI (T.R.G.) and HFSP (U.B.-D.). Research in the the laboratory of U.B.-D. is supported by the Azrieli Foundation, the Richard Eimert Research Fund on Solid Tumors, the Tel-Aviv University Cancer Biology Research Center, and the Israel Cancer Association.

Author information

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Authors

Contributions

U.B.-D. conceived and supervised the project; O.M.E. and U.B.-D. collected the data and performed the computational analyses; V.R., M.A. and U.B.-D. carried out the experiments; D.L. and D.D. assisted with the L1000 assay; S.P. and N.C. assisted with western blots; J.H. assisted with the mutation data analysis; S.P., J.G.D. and F.V. contributed the matched parental and Cas9-expressing cell lines; A.N. and A.T. assisted with the Oncopanel assay and analysis. O.M.E., V.R., R.B., T.R.G. and U.B.-D. analyzed the data and wrote the manuscript. R.B., T.R.G. and U.B.-D. directed the project.

Corresponding author

Correspondence to Uri Ben-David.

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Competing interests

J.G.D. consults for Tango Therapeutics, Foghorn Therapeutics, and Pfizer. T.R.G. is a paid advisor to GlaxoSmithKline and Sherlock Biosciences. R.B. owns shares in Ampressa and receives grant funding from Novartis. D.D. is an employee of Cellarity.

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Extended data

Extended Data Fig. 1 Cas9 introduction activates the p53 pathway (related to Fig. 1).

a, Unsupervised hierarchical clustering of 165 WT/Cas9 cell line pairs, based on their median L1000 transcriptional profiles (landmark space, n = 978 genes). Cell line pairs are colored in red and black, alternately, to highlight that all Cas9 lines cluster together with their parental WT lines. b, Transcriptional activity scores (TAS)6 comparison of technical replicates of 165 parental lines, 165 technical replicates of Cas9 lines, 165 Cas9 lines vs. parental lines, or 22 control vector lines vs. parental cell lines. *, P < 2 × 10–16, P < 2 ×10–16 and P = 2.5 × 10–7, two-tailed paired t test. Data points represent cell line pairs. c, Lack of correlation between Cas9 activity levels (measured by GFP levels; see Online Methods) and the strength of the transcriptional response (measured by TAS). P = 0.68, two-tailed test for association using Spearman’s rho. 158 lines are colored by their TP53 mutation status; 7 lines excluded due to lack of Cas9 activity data. d, The proportion of lines (n = 165) with an activated p53 pathway activity following Cas9 introduction, in TP53-WT vs. TP53-mutant cell lines. *, P = 0.0007, two-tailed Fisher’s exact Test. e, The proportion of TP53-WT lines (n = 61) with an activated p53 pathway activity following Cas9 or empty/reporter vector introduction. *, P = 0.006, two-tailed Fisher’s exact Test. f, The degree and significance of enrichment of the 50 MSigDB Hallmark biological pathways, following the introduction of empty vectors, reporter vectors and Cas9 into TP53-WT cell lines, and the introduction of Cas9 into TP53-mutant cell lines. Black, significantly enriched (GSEA enrichment score with multiple hypotheses correction; q < 0.05) pathways. Orange, the p53 pathway. Each plot represents the results of one Meta expression signature (see Online Methods). g, Comparison of Cas9 activity levels and TAS, as in (d), but only 40 available TP53-WT lines are presented. Cell lines are colored by whether their gene expression profiles were enriched for the p53 Hallmark gene set (and in which direction). P = 0.30, two-tailed test for association using Spearman’s rho.

Extended Data Fig. 2 Confirmation of p53 activation following Cas9 introduction (related to Fig. 1).

a, Left: confirmation of p53 pathway activation in BT159 cell lines by RT-qPCR analysis of 7 transcriptional targets of p53. *, P = 0.017, **, P = 0.0065, ****, P < 0.0001, one-tailed t test. Data values represent the means of 3 replicates, with error bars corresponding to S.D. Right: the average activation of p53 transcriptional targets. P = 0.08, two-tailed one-sample t test. Data values represent the means of the 7 targets, with error bars corresponding to S.D. b, Left: RT-qPCR analysis of 7 transcriptional targets of p53 in A549 (TP53-WT) before and after its transduction with Cas9 or with three control vectors: luciferase, GFP or DNA barcode. *, P = 0.048, one-tailed t test. Data values represent the means of the 3 control vectors and of 3 biological replicates of Cas9, with error bars corresponding to S.D. Right: the average activation of p53 transcriptional targets. *, P < 0.05, two-tailed one-sample t test. Data values represent the means of the 7 targets, with error bars corresponding to S.D. c, Protein levels of Cas9, p53, p21 and a housekeeping protein in HCT116 cells transfected with GFP, Cas9 or a backbone-matched empty vector (EV). Results represent a single experiment. d, Protein levels of Cas9, p53, p21 and a housekeeping protein in isogenic TP53-WT (P) and TP53-null HCT116 cells before and after transduction of Cas9 (C) or of a backbone-matched control vector (EV). Results represent a single experiment. e, Left: RT-qPCR analysis of 7 transcriptional targets of p53 shows p53 pathway activation specifically in the Cas9-expressing TP53-WT HCT116 cells. Data values represent the means of 2 replicates, with error bars corresponding to S.D. Right: the average activation of p53 transcriptional targets. *, P = 0.028, ***, P = 0.0004, ****, P < 0.0001, two-tailed one-sample t test. Data values represent the means of the 7 targets, with error bars corresponding to S.D. Source data

Extended Data Fig. 3 Cas9 introduction activates the DNA damage response (related to Fig. 2).

a, The proportion of cell lines (n = 165) with a positively enriched DNA damage transcriptional signature, following Cas9 introduction. *, P = 0.07; two-tailed Fisher’s exact Test. b, Fluorescent microscopy images of 𝛾H2AX foci (green) and DAPI (blue) in parental TP53-WT HCT116 cells and following Cas9 transduction. Cells with > 5 foci have been marked in white. Scale bar represents 10 µm. c, Quantification of 𝛾H2AX foci from three independent repeats; n = 1,765 and n = 2,523, for WT and Cas9 HCT116 cells, respectively. **, P = 0.0095; one-tailed t test. Data show means, with error bars corresponding to S.D.

Extended Data Fig. 4 Cas9 introduction selects for inactivating TP53 mutations (related to Fig. 3).

a, Unsupervised hierarchical clustering of 42 WT/Cas9 cell line pairs across 40 independent cell lines, based on their genetic profiles. Cell line pairs are colored in red and black, alternately, to highlight that all Cas9 lines cluster together with their parental WT lines. b, The count of overall mutations detected across the 42 WT/Cas9 cell line pairs. c, The number of recurrent COSMIC mutations that differ between the Cas9 lines and their matched WT lines (that is, detected either in the parental or in the Cas9 line, but not in both). Emerging mutations are shown in black, disappearing mutations in gray, for the 25 cell lines with any COSMIC mutations present. *, P = 0.027, one-tailed paired t test. d, Sequencing coverage of the TP53 exons in the three cell line pairs in which emergence or expansion of TP53 mutations were detected. e, Cancer genes ranked by their tendency to acquire mutations in the Cas9 lines. Emerging mutations are shown in black, disappearing mutations in gray. TP53 is highlighted in orange. f, The number of non-silent mutations that differ between WT lines and their reported or barcoded derivatives. No mutation in TP53 was observed in 9 independent experiments across three TP53-WT cell lines. g, Cancer genes ranked by the proportion of silent mutations out of all emerging (silent and non-silent) mutations. TP53 is highlighted in orange, and is among the top ~1% of genes (out of 128 genes with a non-silent mutation present).

Extended Data Fig. 5 Proposed workflow for Cas9-related laboratory experiments.

When conducting systematic CRISPR/Cas9-mediated screens or focused studies in TP53-WT cancer cell lines, we recommend determining the basal activation level of the p53 pathway in the Cas9-expressing line. If there is p53 activation, it is recommended to assess Cas9-derived ongoing DNA damage accumulation as well. Finally, as continuous Cas9 expression poses a selection pressure that over time may be reflected in the emergence or expansion of p53-inactivating mutations, it is recommended to avoid extensive passaging and culture bottlenecks that may accelerate this process.

Supplementary information

Supplementary Information

Supplementary Notes 1–9 and Fig. 1

Reporting Summary

Supplementary Data

Supplementary Data 1–7

Source data

Source Data Fig. 1

Unprocessed Western Blots shown in Fig. 1. Uncropped Western Blot images shown in Fig. 1d,g. The dotted square area indicates which samples were cropped and shown in the main figure.

Source Data Extended Data Fig. 2

Unprocessed Western Blots shown in Extended Data Fig. 2. Uncropped Western Blot images shown in Extended Data Fig. 2c,d. The dotted square area indicates which samples were cropped and shown in the main figure.

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Enache, O.M., Rendo, V., Abdusamad, M. et al. Cas9 activates the p53 pathway and selects for p53-inactivating mutations. Nat Genet 52, 662–668 (2020). https://doi.org/10.1038/s41588-020-0623-4

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