Like baby humans, baby cells don't know for sure what they will be when they grow up. They might mature into a heart cell, a muscle cell or something else entirely.
Ultimately, cell type is dictated by genetics — to become a brain cell, specific brain-cell genes must activate. The same goes for bone cells, blood cells and all other cells that make up various tissue types. And once a cell commits to a single "fate," there's often no switching.
But now, Stanford bioengineer Stanley Qi, PhD, and his team have devised a technique that renders cell identity a little more flexible.
With this tactic, which is a form of CRISPR-Cas9 gene editing, Qi has been able to morph skin cells into brain cells with high efficiency, identifying 74 new genes that potentially govern this transformation. The study appears in Cell Stem Cell.
Qi's method tweaks the original gene-editing purpose of CRISPR. Rather than altering the DNA sequence, it toggles the "on" switch of genes, activating their expression. The technique is called CRISPR-activation, or CRISPRa, and it works by ferrying molecular guides and associated “activator proteins” to specific genes to activate them.
Past methods that convert one cell type into another do so with a spotty rate of success — in the best case, transforming skins cells to neuron cells typically work for less than 30 percent of cells. Using CRISPRa, the efficiency can reach 80 to 90 percent.
"The previous CRISPR methods have largely focused on manipulating one gene in one cell, but we expanded on that, because in a cell's developmental process, it's really a coordination of multiple genes that work together and allow for cell identity," said Qi. "The power of CRISPR is that we can use it to flexibly deliver more than one gene-activation agent, so by doing that we try to find synergy between these genes that show the best combinations to prompt a specific cell type."
Qi and colleagues started by sorting out which genes were the most likely to promote neuronal growth. Using a screening technique, the researchers examined all transcription factors, which serve as master controllers of cell fate. From thousands of transcription factors, Qi and his team defined 74 that, when turned on, could generate the physical neuron cell types.
Investigating further, the scientists chose 20 factors that seemed to have the most potential for creating neurons and subsequently delivered activated versions of those genes — one gene per cell — this time looking to see if the genes changed the cells' molecular nature.
Indeed they saw the neuron molecular "signature" after sequencing the cells’ genome. Paired with the success of a test called a "patch clamp" experiment, which verifies that the neuron can fire quintessential electrical signals called action potentials, Qi felt confident that what they were seeing were neurons, and not some other neuron-like imposter.
“CRISPR-based screen techniques have been used to study cancer cell growth and death, and to our knowledge, this is the first time it’s been used to reveal factors that control cell fate,” said Yanxia Liu, PhD, postdoctoral scholar and lead author on the study.
The CRISPRa method doesn’t currently offer an immediate therapy, but its ability to thoroughly identify the genes that underpin specific cell types is a crucial advance in the regenerative medicine field. It's also a step that could be applied to other cells, not just neurons.
"I could imagine doing this with bone cells, heart cells or muscle cells," said Qi. "We're hoping that we can use CRISPRa to transform skin cells to other types of therapeutic cells more easily and cheaply. That would be very exciting."
Photo by ZEISS Microscopy