What Is CRISPR and CRISPRi?

The gene-editing technique known as CRISPR has become the darling of the laboratory world, most recently garnering its discoverers a Nobel Prize. The method is also taking early steps into the clinic as the basis for experimental gene therapies, for example, for a genetic form of blindness.

Several years ago, researchers at the University of California, San Francisco, debuted a variation of CRISPR that lets scientists switch off — and back on — specific genes. Dubbed CRISPR interference, or CRISPRi, this technique is different from classic CRISPR in that it’s temporary and reversible — no permanent changes are made to the DNA.

Now, one of CRISPRi’s creators, UCSF researcher Martin Kampmann, Ph.D., has teamed up with researchers from the Allen Institute for Cell Science, a division of the Allen Institute, to release a human stem cell line that contains CRISPRi for dialing down the activity of different genes. This publicly available cell line was made by gene-editing human induced pluripotent stem cells, naïve adult human cells with the potential to give rise to many other kinds of tissues, and will allow any research lab to use the technique for their own discoveries.

“This is really the next generation of knock-downs,” said Ruwanthi Gunawardane, Ph.D., Deputy Director and Director of Stem Cells and Gene Editing at the Allen Institute for Cell Science, who led the development of the publicly available CRISPRi cell line. Knock-downs refers to the general lab technique that temporarily reduces a gene’s activity to better understand what that gene does under normal conditions.

Kampmann, who is also an Allen Distinguished Investigator studying the cellular underpinnings of Alzheimer’s disease, uses the technique to switch off certain genes in neurons. He and his colleagues, including Dr. Michael Ward at the NIH, developed CRISPRi in stem cells, naïve cells that have the potential to grow into several other types of cells, like neurons or muscle cells. In recent years, human stem cells taken from adult volunteers have risen in popularity for cell biology research, but existing methods for “knocking down” genes didn’t work very well in these cells. Cell biologists needed a new approach.

This is really the next generation of knock-downs.”

— Ruwanthi Gunawardane, Ph.D.

Broadly speaking, CRISPR works by pairing molecular “scissors,” a protein called Cas9, with a guide sequence that sticks to its mirror piece of DNA like two sides of a zipper. That molecular guide allows the Cas9 scissors to cut the DNA at a precise spot. The technique has enabled researchers to make specific genetic changes, either by disabling an entire gene or by introducing a new DNA sequence along with the cutting tool.

In CRISPRi, Kampmann and his colleagues figured out a way to disable the scissors protein, Cas9, and attach instead a protein that blocks normal gene activity. The end result is a modified CRISPR that dampens gene activity without editing the DNA itself.

“You now have a way to basically turn the knob on how actively a specific gene is expressed,” or switched on, said Kampmann. “This is a great way to understand how individual genes work, by switching them on and off. But you can also use it broadly to study a cellular function you’re interested in.”

Kampmann used the technique to understand which genes are essential for neurons to survive and deal with stress. He and his colleagues used CRISPRi to systematically screen through and switch off each of approximately 20,000 genes encoding proteins in human stem cells that they then spurred to develop into neurons. The technique of genetic screening is like a detective mystery. In this case, Kampmann and his team were looking for factors that controlled neuronal survival and response to oxidative stress, which neurons experience in aging and neurodegenerative diseases.

Because adult neurons don’t divide, in the brain or in a petri dish, it’s impossible to do this kind of assay without the use of stem cells. Next, they want to use the technique to find genes involved in brain diseases like Alzheimer’s and ALS.

The Allen Institute Collaboration

Through visits to the Allen Institute, Kampmann learned about the work Gunawardane and her team were conducting to develop gene-edited, fluorescently tagged human stem cell lines for their own research and for the broader scientific community. The two groups teamed up to develop a CRISPRi cell line that anyone could use.

Gunawardane and other Allen Institute for Cell Science researchers are using the technique themselves to study genes important for certain structures in the nucleus, the information storage center of our cells. In this case, they’re studying the genes’ activity directly in stem cells, but the potential applications are broad, Gunawardane said. Their studies of the nucleus are aimed at understanding basic biology, but dysfunctions in certain nuclear structures are associated with diseases such as progeria, which accelerates aging.

Researchers could also use CRISPRi to screen for genes involved in a specific disease, as Kampmann is doing with brain diseases, or cellular functions connected to disease — such as DNA damage repair, a cellular process that seems to prevent tumor formation.

“You could use this for any screen you can think of in stem cells or cell types differentiated from those stem cells,” Gunawardane said.