Paper of the month august 2018

CRISPR-Cas9 genome editing induces a p53-mediated damage response

Human pluripotent stem cells (hPSCs) represent a great opportunity for both basic research and therapy development. CRISPR-Cas9 offers a valuable tool for hPSCs engineering, however so far genetic engineering in those cells has showed a lower efficiency compared to the application of CRISPR to other cell types. In this paper, the authors aim to uncover the reason of the inefficient gene engineering in hPSCs. To this end, they develop a platform that allows for solid gene editing. This is represented by hPSCS cell stably expressing an inducible Cas9 – from now on defined as iCas9- under the control of a doxycycline dependent promoter. Which means that only upon doxycline supplementation the Cas9 will be expressed. As first experiment they target different genes by supplying both doxycline – thereby inducing the iCas9- and a gRNA library. Compared to the control, they could achieve nearly 98% of target cleavage. However, the viability of the cells was dramatically reduced. To test if the result was target dependent, they decide to target the MAPT gene, which is known to not have any vital function for the cells. Nevertheless the result was the same: high cleavage, low viability.

To rule out the possibility that this was due to the elevated Cas9 expression, they decided to supply the Cas9 as protein, which has a shorter half-life because is degraded by the cell´s proteasome system. Once again, there was no change in the result. At this point the idea the cleaving the DNA may be toxic for the cells starts taking place. They decide then to use again the iCas9 but this time they use two different gRNA library: one is designed to target different the genes, the other is no able to guide the Cas9 on the target and thereby no DNA cut is induced. The results showed that the cells in which no DNA cleavage occurred show a good viability and this is independent of the Cas9 expression. The scientists decide then to use bioinformatics tools – in particular Transcriptome analysis – to identify which genes may be involved this high mortality in the hPSCs. They identify p53 as a key player in this event. In fact in the following experiment they proved that if p53 is knocked out – which means that is no functional anymore – the cells - in which a double stranded break was induced – are still vital. Importantly it is showed that the increased cell deaths in target independent, which means that just cutting the DNA – no matter the sequence – is toxic in this cell type.

All together the results show that Cas9 induced double stranded break can lead to a toxic response p53 mediated. p53 is a known oncosopressor gene, defined as the “cell guardian” and involved in several pathways ranging from the cell-cycle check points skipping control to aberrant cell metabolism detection, which are indicative of a cancerogenous behaviour. CRISPR based therapy relies on the efficient cells engineering. The cells after the treatment –before being injected in to the patient – are enriched to a sufficient number for effective treatment. However, the results showed pose a question mark about the p53 status in the cells surviving the CRISPR treatment and in the overall safety of this technique. In fact as showed by the authors, hPSCs were able to survive when p53 was mutated or knocked out. This means that during the gene engineering process we may indirectly select for cells that have a mutation in p53. As previously said, p53 is critical to assure control on the homeostasis of the cells, in particular preventing aberrant cancer. Thereby when it comes to therapeutic applications, gene engineered cells should be checked for p53 mutation before and after the process, to avoid to inject p53 mutated cells that may be highly dangerous for the patient.