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Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance

Anne Ramsay Bowden, David A. Morales Juarez, Matylda Sczaniecka-Clift, Maria Martin Agudo, Natalia Lukashchuk, John C. Thomas, Stephen P. Jackson

Posted on: 27 April 2020

Preprint posted on 20 February 2020

Article now published in eLife at http://dx.doi.org/10.7554/eLife.55325

CRISPR-Cas9 screens and catch p53

Selected by Mila Basic

Categories: biochemistry, cell biology

 

Background

CRISPR-Cas9 has completely transformed the field of high-throughput genetic screens. The vastly reprogrammable, modular, and easily scalable nature of CRISPR-Cas9 design triumphed over other genome editing tools, or RNAi-based gene silencing methods [1], [2]. As such, CRISPR-Cas9 genetic screens are increasingly implemented as a basic method in many labs. Recently, a study by Haapaniemi et al. raised a concern connected to the Cas9 mechanism of action, and if it negatively impacts the results of the screens it is used for [3]. Specifically, to efficiently edit the DNA, Cas9 induces a double stranded break (DSB) [4]. DSBs are great threats to genetic stability, and cells have developed multiple pathways to minimize their impact. Namely, DSBs have been shown to induce p53-mediated cell cycle arrest [5], [6], or p53-mediated apoptosis [7], [8]. The obvious question arose – what is the impact of p53 on CRISPR-Cas9 genetic screen performance? How do protective properties of p53 in edited cells potentially impair their proliferation or viability, hence, reliability of screen results?

 

Key Findings

In order to determine the effect of p53 on CRISPR-Cas9 genetic screens, the authors carried out two parallel screens, one in wild type (WT), and one in p53 knockout (KO) retinal pigment epithelial cells. The custom-made library included guides targeting 852 DNA damage response genes, 112 olfactory receptor genes (serve as non-essential gene control), and 14 sequence-scrambled negative controls. Upon performing the screen, they established that the depletion of core essential genes (defined by [9]) was clearly detectable in both p53 WT and p53 KO background, although the p53 KO screen slightly outperformed the WT screen. Interestingly, while log fold changes were not significantly different for non-essential genes, for essential genes they were indeed significantly lower in p53 KO compared to WT screen. This could unexpectedly be attributed to p53-mediated proliferation defect leading to an attenuated gene depletion impact, hence, narrower guide distribution. In addition, they detect differences for guides targeting p53 itself, and positive or negative regulators of p53 between the genetic backgrounds. In spite of confirming p53 effect on screen performance, the authors discuss that reliable data can nonetheless be obtained from p53 WT cells. This is further strengthened by comparative analysis of 5 other screens from p53 WT cells, including aforementioned Haapaniemi et al. Similarly to what Brown et al. from University of Toronto [10] found as well – p53 matters, but does not necessarily impair CRISPR-Cas9 genetic screening.

 

What I like about this preprint

This preprint caught my attention because the question of p53 impact on CRISPR-Cas9 screens was regularly raised in discussions at my institute. Despite being outside of the field, I chose to highlight it because I really liked the authors’ systematic approach to answering the question of p53 impact on CRISPR-Cas9 screens. In addition to dissecting multiple aspects of how exactly p53 negatively effects screen results, they nicely demonstrate how this can be compensated for, and minimized. It serves as an excellent rule of thumb for optimal screen design in p53 WT cells.

 

Open questions

What would be the key consideration when designing a screen where the library size is significantly bigger than a thousand genes? If guide representation has to be compromised due to library size, is it still reasonable to use p53 WT cells?

Why is p53-mediated response to Cas9-induced DSBs not more prominent when it comes to non-essential genes, as compared to essential genes?

What is the contribution of dual guide design compared to a single guide system to the response between the genetic backgrounds? Did an increased number of DSB per cells affect the screen outcome in p53 WT cells?

How do these findings apply to cells where p53 is mutated, like some cancer cell lines? Can reliable screens be performed in them? What are some aspects that need to be taken into consideration?

 

References

[1]  D. Carroll, “Genome Engineering with Targetable Nucleases,” Annu. Rev. Biochem., vol. 83, no. 1, pp. 409–439, Jun. 2014.

[2]  M. Boettcher and M. T. McManus, “Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR,” Mol. Cell, vol. 58, no. 4, pp. 575–585, 2015.

[3]  E. Haapaniemi, S. Botla, J. Persson, B. Schmierer, and J. Taipale, “CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response,” Nat. Med., vol. 24, no. 7, pp. 927–930, 2018.

[4]  M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science (80-. )., vol. 337, no. 6096, pp. 816 LP – 821, Aug. 2012.

[5]  A. Di Leonardo, S. P. Linke, K. Clarkin, and G. M. Wahl, “DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts.,” Genes Dev. , vol. 8, no. 21, pp. 2540–2551, Nov. 1994.

[6]  S. P. Linke, K. C. Clarkin, A. Di Leonardo, A. Tsou, and G. M. Wahl, “A reversible, p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage.,” Genes Dev. , vol. 10, no. 8, pp. 934–947, Apr. 1996.

[7]  N. D. Lakin and S. P. Jackson, “Regulation of p53 in response to DNA damage,” Oncogene, vol. 18, no. 53, pp. 7644–7655, 1999.

[8]  A. R. Clarke et al., “Thymocyte apoptosis induced by p53-dependent and independent pathways,” Nature, vol. 362, no. 6423, pp. 849–852, 1993.

[9]  T. Hart et al., “Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens,” G3 Genes|Genomes|Genetics, vol. 7, no. 8, pp. 2719 LP –2727, Aug. 2017.

[10]  K. R. Brown, B. Mair, M. Soste, and J. Moffat, “CRISPR screens are feasible in TP53 wild-type cells,” Mol. Syst. Biol., vol. 15, no. 8, p. e8679, Aug. 2019.

Tags: crispr-cas9, genetic screens, p53

doi: https://doi.org/10.1242/prelights.19433

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Author's response

John C. Thomas and Stephen P. Jackson shared

What would be the key consideration when designing a screen where the library size is significantly bigger than a thousand genes? If guide representation has to be compromised due to library size, is it still reasonable to use p53 WT cells?

If guide representation is compromised, then using p53 WT cells would exacerbate the problem and therefore we’d recommend the researcher to consider whether using p53 WT cells is absolutely required, and if so to increase the number of infected cells if possible.

Why is p53-mediated response to Cas9-induced DSBs not more prominent when it comes to non-essential genes, as compared to essential genes?

The strength of the log fold change observed is dependent on the relative essentiality of the gene, compared to the rest of the genes covered by the library. So, for example, if all the genes targeted by a CRISPR library halved the growth rate of cells when knocked out, then you would find that all guides resulted in a final log fold change of around zero, as all the cells were equally affected. We believe that WT p53 impacts the growth of all cells in a CRISPR screen by responding to DNA double-stranded breaks, reducing the observable additional effect caused by knocking out specific genes. This difference is more visible in essential genes because they have a greater differential effect on cells’ growth.

What is the contribution of dual guide design compared to a single guide system to the response between the genetic backgrounds? Did an increased number of DSB per cells affect the screen outcome in p53 WT cells?

We speculate that because the dual guide system should generate twice the number of DNA double-strand breaks per cell compared to a single-guide system, it will be even more sensitive to p53 status than single-guide approaches. This would be an interesting avenue for further exploration.

How do these findings apply to cells where p53 is mutated, like some cancer cell lines? Can reliable screens be performed in them? What are some aspects that need to be taken into consideration?

We expect that our findings will be widely relevant to various cell lines, including cancer cell lines with TP53 mutations. Work from us and others has established that p53 deficient cancer cell lines can indeed be successfully used in CRISPR-Cas9 screens. Factors we highlight, including guide representation and sequencing depth, remain important considerations in screen design in all cellular backgrounds.

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