As a tool for editing cell DNA, the protein Cas9 is prized for its precision, its ability to snip DNA at specific locations without producing repercussions elsewhere in the genome. On the basis of a recent study by Dana-Farber scientists, that reputation for unobtrusiveness will have to be revised.
In a paper in Nature Genetics, the researchers report that Cas9 — the “molecular scissors” used by scientists around the world to add, remove, or alter genetic material — activates the TP53 gene in cells. Known as the master regulator of the genome, TP53 plays a key role in repairing damaged DNA. It also is notorious as the most frequently mutated gene in cancer.
The finding means that scientists using CRISPR-Cas9 — the gene-editing system based on Cas9 — will need to take this TP53-activating ability into account when designing experiments and interpreting findings, the study authors say.
“A wide range of studies have explored how cell lines used in laboratory research can change over time. These changes can create discrepancies between one lab’s findings and another’s and introduce experimental errors,” says study co-author Rameen Beroukhim, MD, PhD, of Dana-Farber and the Broad Institute of Harvard and MIT. “CRISPR-Cas9 has become one of the most important systems for the study of cancer and cell biology in general. But it hasn’t been known whether Cas9 itself results in changes in gene activity.”
CRISPR-Cas9 borrows from a natural defense system used by bacteria. Bacteria cells maintain a collection of DNA segments from viral invaders — a molecular archive of the viruses they’ve encountered. Should these viruses, or similar ones, attack again, bacteria use these segments — assembled into CRISPR arrays — to quickly produce RNA strands that target the invaders’ DNA. Cas9 or similar enzymes then converge on the RNA targets and chop the DNA apart, disabling the virus.
In CRISPR-Cas9, researchers introduce Cas9 to a cell, followed by a specially made segment of RNA. As in bacteria, the RNA goes to a specific site on the genome and prompts Cas9 to cut the DNA at that spot. Researchers use the technique to create gene mutations or splice in or snip out genetic material to explore the effect on cells.
In the new study, researchers introduced Cas9 to 165 cancer cell lines and found that it ramped up the activity of the p53 pathway — the series of DNA-repair proteins switched on by the TP53 gene. The upregulation of p53 caused cell growth and division to slow because cells were devoting more energy and resources to DNA repair. An analysis of 42 of the cell lines showed that the slowdown was often temporary: lines treated with Cas9 often developed mutations that inactivate p53, allowing them to grow more quickly.
The researchers also found that activation of the p53 pathway by Cas9 affected how cells responded to chemical agents such as drug molecules and to genetic manipulations such as those performed with CRISPR-Cas9. The upshot is that researchers cannot assume that Cas9 has no impact on cell functioning beyond its role in cutting DNA.
While researchers have not yet discovered the mechanism that links Cas9 to an uptick in p53 activity, the implications for research involving CRISPR-Cas9 are clear.
“When conducting these experiments, it’s important to carefully investigate p53 levels in cells following the introduction of Cas9,” says Dana-Farber’s Veronica Rendo, PhD, the co-lead author of the study with Oana Enache of the Broad Institute. The study’s senior author is Uri Ben-David, PhD, a former postdoctoral fellow in Beroukihm’s lab now at Tel Aviv University. “We’ve shown that some of the changes in cell behavior following the use of CRISPR-Cas9 may result from Cas9 itself, rather than the specific genetic manipulations performed in the experiment. Those changes need to be considered in experimental design and data analysis.”