CRISPR-Cas9 and p53

The CRISPR-Cas9 system represents a remarkable breakthrough in genome editing technology. With relative ease and amazing precision, investigators may now alter or replace genes in the genomes of organisms across the evolutionary spectrum. The potential for human disease treatment or prevention is incredible – an observation not lost on the pharmaceutical companies that have invested heavily in the approach.

All the excitement notwithstanding, some problems have arisen with the technology that may limit its usefulness going forward. Specifically, two papers from the journal Nature Medicine[1],[2] suggest that the “genome guardian” protein, p53, is activated in response to employment of CRISPR-Cas9. To understand what p53 is responding to, a brief description of how the CRISPR-Cas 9 system works is warranted.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR associated protein 9) system employs specific enzymes and carefully designed RNA guides to seek out distinct genomic DNA sequences for editing, removal, or replacement. The process involves the cleavage of DNA – an action not unnoticed by the human cell. Indeed, it is at this step that the cell takes exception to indiscriminate cutting of its DNA and turns on the (DNA) damage response pathway – orchestrated by p53. The aforementioned papers document the effects in human cells of such initiation of the p53-mediated reaction to DNA strand breaks.

CRISPR Cas9 system (Source: Marius Walter; Wikimedia Commons)

In the paper by Haapaniemi and colleagues, the authors observe that CRISPR-Cas9 activates the p53 pathway in human cells, and limits the efficiency of the gene editing process. In contrast, inhibiting the damage response pathway allows for greater effectiveness of the CRISPR-Cas9 genomic modification apparatus. The obvious problem is that any attempts to down-regulate p53, or to select for cells that have reduced activity of the pathway, by definition create cells potentially susceptible to cancerous transformation. The authors suggest that in future work, mechanisms by which the DNA repair pathway could be modulated – perhaps turned off during gene editing, but reinitiated shortly thereafter, represent an appropriate work around.

In the study by Ihry and colleagues, attempts are made to employ CRISPR-Cas9 to reprogram pluripotent stem cells. Once again, the gene editing procedure elicits a p53-mediated DNA damage response, and the cells undergo programmed cell death (~apoptosis). Cells that survive are very much at risk as they may harbor down-regulated p53 – potentiating the possibility of an accumulation of offsite mutations. As above, the answer seems to point to modulating p53 activity – turning it off for the editing step, but reactivating immediately following.

Much has been written about these results, both in the scientific literature and popular press. No one is calling for the technique to be abandoned; rather, additional research will be required before it can be expected that the human cell’s sophisticated and evolutionarily developed capacity for protecting itself can be sidestepped. Expect a wealth of research to be published on the topic in coming months. Stay tuned!

SRT – August 2018

[1] E. Haapaniemi et al., Nat Med. (2018) doi: 10.1038/s41591-018-0049-z. [Epub ahead of print] (Link to Text)

[2] R.J. Ihry et al., Nat Med. (2018) doi: 10.1038/s41591-018-0050-6. Epub 2018 Jun 11. (Link to Text).