In the new study, the MIT team deactivated Cas9’s cutting ability and engineered the protein so that after binding to a target site, it recruits transcription factors (proteins that are required to turn genes on).
By delivering this version of Cas9 along with the guide RNA strand into single cells, the researchers can target one genetic sequence per cell. Each guide RNA might hit a single gene or multiple genes, depending on the particular guide sequence. This allows researchers to randomly screen the entire genome for genes that affect cell survival.
“What we decided to do was take a completely unbiased approach where instead of targeting individual genes of interest, we would express randomized guides inside of the cell,” Lu says. “Using that approach, can we screen for guide RNAs that have unusually strong protective activities in a model of neurodegenerative disease.”
The researchers deployed this technology in yeast cells that are genetically engineered to overproduce a protein associated with Parkinson’s disease, known as alpha-synuclein. This protein, which forms clumps in the brains of Parkinson’s patients, is normally toxic to yeast cells.
Using this screen, the MIT team identified one guide RNA strand that had a very powerful effect, keeping cells alive much more effectively than any of the individual genes that have been previously found to protect this type of yeast cell.
Further genetic screening revealed that many of the genes turned on by this guide RNA strand are chaperone proteins, which help other proteins fold into the correct shape. The researchers hypothesize that these chaperone proteins may assist in the proper folding of alpha synuclein, which could prevent it from forming clumps.
Other genes activated by the guide RNA encode mitochondrial proteins that help cells regulate their energy metabolism, and trafficking proteins that are involved in packaging and transporting other proteins. The researchers are now investigating whether the guide RNA turns on each of these genes individually or whether it activates one or more regulatory genes that then turn the others on.