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p53 DNA Damage Response Causes Concern for CRISPR–Cas9 Genome Editing

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 Review of “CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response” and “p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells” from Nature Medicine by Stuart P. Atkinson

As CRISPR–Cas9 genome editing holds the potential to completely revolutionize many areas of research, the safety and efficacy of the technology come under a reasonable level of scrutiny. While one study suggesting off-target effects dampened the fervor surrounding the field [1], a reevaluation of the study underlined the highly targeted nature of CRISPR–Cas9-mediated genome editing [2]. However, two recent studies published in Nature Medicine [3, 4] now report another cause for concern: the potential for tumorigenesis in cells lacking the p53 tumor-suppressor gene following genome editing.

The first study [3], led by Bernhard Schmierer and Jussi Taipale (Karolinska Institute, Stockholm, Sweden), studied CRISPR–Cas9 genome editing in hTERT-immortalized human retinal pigment epithelial cells (RPE1) to identify essential genes [5]. Haapaniemi et al. discovered that genome editing and the subsequent heightened presence of DNA double-strand breaks (DSBs) induced a p53-mediated DNA damage response (DDR) and cell cycle arrest, thereby causing the low-efficiency editing rate observed in RPE1 cells. However, the inhibition of proper p53 function prevented the DDR and increased CRISPR-Cas9 editing efficiency. While this seems like an efficient way to promote genome editing, p53 inhibition may transiently permit cells to accumulate tumorigenic alterations, while cells with dysfunctional p53 may be preferentially edited compared to “normal” cells, thereby propagating a potentially tumorigenic genome edited cell population. 

The second study [4], led by Ajamete Kaykas (Novartis Institutes for Biomedical Research, Cambridge, MA, USA), assessed genome editing in human pluripotent stem cells (hPSCs) in the hope of uncovering a means to increase editing efficiency. Ihry et al. consolidated a two-component Cas9 system into a single adeno-associated virus integration site 1 (AAVS1) targeting vector that permitted doxycycline-induced Cas9 expression. Excitingly, this approach led to an average insertion or deletion (indel) efficiency greater than 80%; however, the Cas9-induced high indel generation and associated DSB levels induced P53/TP53-dependent toxicity and significant death of hPSCs. The authors note that as hPSCs can acquire P53 mutations [6], abnormal hPSCs may represent the dominantly editing cell, potentially causing safety problems for cell replacement therapies.

While these studies represent a concern for genome editing of both normal and pluripotent stem cells, the CRISPR-Cas9 death knell has yet to sound! Instead, these results will help us to design new strategies and safety protocols that will ultimately boost the safe and effective application of genome editing as we move forward.

For more on CRISPR-Cas9 safety and effectiveness, stay tuned to the Stem Cells Portal!

References

  1. Schaefer KA, Wu W-H, Colgan DF, et al., Unexpected mutations after CRISPR–Cas9 editing in vivo. Nature Methods 2017;14:547.
  2. CRISPR off-targets: a reassessment. Nature Methods 2018;15:229.
  3. Haapaniemi E, Botla S, Persson J, et al., CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nature Medicine 2018;24:927-930.
  4. Ihry RJ, Worringer KA, Salick MR, et al., p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. 2018;24:939-946.
  5. Luo M and Chen Y, Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future. Int J Ophthalmol 2018;11:150-159.
  6. Merkle FT, Ghosh S, Kamitaki N, et al., Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 2017;545:229.