Researchers discover what they didn’t set out to find.

By Jeffrey M. Perkel

 

It’s not often one literally gets to see history made in the scientific arena. But that’s what happened in the summer of 2012, in the pages of Science magazine.

Jennifer Doudna of the University of California, Berkeley, and Emmanuelle Charpentier, then at Umeå University in Sweden, demonstrated the molecular mechanics underlying a quirky bacterial system called CRISPR/Cas. More to the point, they demonstrated that the system could be reprogrammed to do something incredibly useful, genetically speaking: a process called genome editing.

That such a system exists naturally and can be harnessed for the good of the research community, seems almost incredible. As Harvard University geneticist George Church, who uses the system, told me in August, “It’s a real gift from biology.” But it also is proof of the power of basic biological research, as the power of CRISPR/Cas is something nobody could have predicted at the outset.

In brief – and I’ll expand on this below – the CRISPR/Cas system gives researchers a tool to make specific, surgical changes in the genome of living cells.

Suppose, for instance, that researchers wanted to repair the genetic defect in an individual with Huntington’s disease. Using the Nobel Prize-winning development known as “induced pluripotent stem (iPS) cells,” they could take cells from this individual and convert them into a kind of embryonic stem cell, which can develop into any kind of cell or tissue in the body. They could then use the CRISPR/Cas system to repair the defect in those cells, convert those cells into neurons and, in theory anyway, transplant them back into the patient.

Now, that’s all a LONG WAY OFF. But that’s the promise of CRISPR/Cas, and it wasn’t lost on the scientific community.

Within months of Doudna and Charpentier’s original paper, the scientific literature was awash in papers extending their findings. Their paper has been cited 78 times, according to the Web of Science, a database that tracks such things – that’s a huge number for just one year. (By comparison, the average paper is cited perhaps once in a year, and even in the high-powered journal Science, a typical paper is cited about 30 times in two years.)

In recognition of the frenzy the discovery has set in motion, Science in August published a nice overview of the research to date, entitled “The CRISPR Craze.”

So, just what is CRISPR/Cas? Well, first of all, it’s a clumsy acronym. CRISPR is short for “Clustered Regularly Interspaced Short Palindromic Repeats;” Cas is “CRISPR-associated.”

Basically, as Church explained it to me, CRISPR/Cas represents a kind of bacterial adaptive immune system, like your B- and T-cells.

At the heart of the system is a series of repeating sequences in the genomes of bacteria. Researchers had for years noticed these funny elements, but had no idea what they might be doing. Then one day, somebody realized they were similar to the genetic material of viruses that infected bacteria. That led to the recognition that when bacteria were infected with a pathogen, they recorded the event in their genetic code, enabling them to better recognize and fight the pathogen in the future.

How do they fight those pathogens? Enter Cas. This is an enzyme that makes cuts in DNA, but not just anywhere. Cas cuts DNA at a sequence specified by the CRISPR elements. The CRISPR sequences are copied into RNA, which the cell then dices up into short pieces; these seek out their matching sequences in the pathogen’s genome, and Cas goes to work, inactivating the invading genetic material.

The DNA cut produced by Cas can be repaired by either of two mechanisms. The simpler one, called non-homologous end-joining, simply tries to suture the two cut ends together again, an error-prone process. The second method, homology-directed repair, uses a backup copy of the gene as a template to fix the damaged copy. Researchers can supply their own template sequences to direct this process to, for instance, insert a gene that would cause the cells to fluoresce whenever a specific gene is turned on.

Now, to be clear there are other systems that do the same thing, particularly zinc finger nucleases (ZFNs) and TALE nucleases (TALENs). These are man-made proteins that can be programmed to target specific DNA elements, no guide RNA required. They have proven to be exceptionally useful research tools, and some are even being tested in clinical trials. But they’re also expensive, tedious, and challenging to build, and not every ZFN or TALEN cuts as expected.

CRISPR/Cas essentially has none of those limitations – anyone can order the protein and a short targeting RNA sequence (a relatively inexpensive proposition), and by all accounts, the process just seems to work.

In their 2012 paper, Doudna and Charpentier’s team demonstrated that Cas uses two short RNAs, called a crRNA and tracrRNA, to work its magic. Those two RNAs could be linked into a single molecule, called a guide RNA, making the system a simple two-component tool.

Then – and this is the important point – they demonstrated they could direct Cas to a sequence of their choosing by adding an appropriate guide molecule.

They did that in a test tube, but it wasn’t long before others demonstrated the process also works in live cells. In January, six papers were published (including one from Doudna’s lab) showing that CRISPR/Cas can make genetic alterations in human cells, mouse cells, bacterial cells, and even in zebrafish.

Since then, other researchers have replicated and extended these findings. Some have shown, for instance, that the system is “multiplexable” – that is, users can make multiple genetic modifications in the same cell by supplying more than one guide RNA, something that isn’t possible with TALENs and ZFNs. (One paper demonstrated it was possible to make five alterations simultaneously.) Others have demonstrated they can use the CRISPR/Cas system, with some judicious protein modifications, to alter the expression of genes, using the system to target genetic regulatory proteins to specific genomic addresses. (That latter point is graphically illustrated at the bottom of the figure above.)

A number of researchers have tackled the question of CRISPR/Cas’s specificity – that is, how likely is it to modify an unintended sequence along with the user’s desired target. (Short answer: The system does have some issues with off-target effects, but they can be addressed using relatively simple tweaks and workarounds; one approach is described here.) Some are exploring the therapeutic applications of the technology. And at least two papers (here and here) have shown that it is possible to make genetic alterations in the iPS cells I mentioned earlier, a precursor to the idea of genetic repair.

It’s a remarkable string of developments in so short a time. And as Doudna said when I spoke with her in March, it arose not from a goal-oriented research project — somebody saying, hey guys, let’s go invent a useful genetic tool! — but basic research, a desire to understand something new and interesting:

“I think it’s a really great example of how fundamental basic research, in this case, which was not aimed at any particular target or goal or certainly a particular application, led to the discovery of a system that may turn out to be a really transformative technology for genome engineering.”

Given the staggering difficulties researchers face getting funding for basic research projects, somebody in Washington would do well to remember that.