In Part 1, we established that the CRISPR/Cas system is a component of adaptive immunity in many types of bacteria which works by cutting DNA – usually belonging to invading viruses – at a position specified by an attached guide RNA molecule. Two scientists, Emmanuelle Charpentier and Jennifer Doudna, initially interested simply in understanding the workings of bacterial RNAs, first showed in a 2012 paper that the Cas9 enzyme could be “re-programmed” by attaching a synthetic RNA, which caused it to search for and cut any DNA with that same code. Though they demonstrated this only in test tubes and not in living cells at the time, the implications were clear. Working at around the same time but published in early 2013, Feng Zhang used a further-modified CRISPR/Cas9 system to make genomic edits in laboratory-cultured mouse and human cells – leading to a still-unresolved patent battle over which of these groups is the real “inventor” of this technology.
Cutting and Pasting
Leaving that aside, we have so far talked only about Cas9’s ability to cut DNA, and detailed its evolutionary origins as a weapon against pathogens – which may leave onewondering how we have managed to co-opt this system for modifying DNA in a non-destructive way. Once again, the answer comes by way of exploiting nature’s inventions: in most organisms, there exist processes for repairing damaged DNA in cells. This happens in two main ways (see diagram below): Non-homologous end joining (NHEJ), in which the cell’s repair enzymes detect the presence of two broken ends of DNA (a double-strand break, or DSB) and simply try to stick them back together, often introducing errors of one or a few base pairs; and homology-directed repair (HDR), in which the cell attempts to repair the gap based on some nearby template carrying homologous (matching) regions to those around the cut site. This means we can artificially introduce DNA molecules carrying long stretches which match the existing genomic sequence (homology arms), encouraging the repair enzymes to use this as a template, and thus inserting any DNA of our choosing into the gap, from a few altered letters to a whole new gene.
From Mice to Mushrooms
This system has proven to be extraordinarily versatile, working in essentially every kind of organism in which it has been tested. Naturally, a great deal of attention has focused on CRISPR’s potential use in people, whether in the treatment of genetic diseases or, more controversially, the creation of designer humans. There are, of course, important ethical questions to explore and significant technical challenges (e.g. off-target effects) still to be overcome before CRISPR can become a real therapeutic tool. However, it has already been used experimentally, for example, to knock HIV infections out of laboratory-cultured cells, to treat muscular dystrophy in mice, and to eliminate a disease-causing mutation in human embryos. In agriculture, too, CRISPR has seen a multitude of applications including crop improvements, mushrooms which stay fresh longer, and even hornless cattle.
A Molecular Swiss Army Knife
However, it is in basic biological research that CRISPR has seen the greatest explosion of uses. Simply being able to readily delete or tinker with specific genes in our experimental systems such as fruit flies or stem cell lines is extremely useful in figuring out basic gene functions. But the Cas9 enzyme itself has turned out to have another very helpful property: its two catalytic activities (binding to a DNA strand, and then cutting it) work completely independently of each other. It is therefore possible to engineer so-called “dead Cas9” with the nuclease activity removed so that it no longer cuts DNA, but simply locates a target site. Different activities can be plugged in – gene activators or repressors including epigenetic modifiers, fluorescent tags such as GFP to visualise the structure of the genome, or modification of single bases (we wrote a previous blog post about that). There are also a range of CRISPR-related enzymes besides Cas9 whose properties are being explored – for example, Cas13 targets RNA as opposed to DNA. This holds particular promise as a therapeutic tool because, though DNA is the long-term storage medium of genomes, in most cases genes need to be transcribed to a transient RNA copy in order to be expressed; therefore, a system allowing modification of the RNA “transcriptome” could permit fine-tuning of the biological actions of genes in a temporary and reversible way, without making risky permanent changes to the DNA copies in the genome.
The Consequences of Curiosity
We’re only beginning to explore the variety of applications emerging from this once-obscure quirk of bacterial biology. If there is a lesson to be drawn from the CRISPR story, it is that major breakthroughs in science often emerge serendipitously and from sources nobody would predict. Of all the researchers who made the critical early discoveries leading to CRISPR, none were searching for ways to edit the human genome, or directly studying human diseases at all. A small community studying the genetics of microbes noticed an inexplicable feature, and explored it primarily on the basis of their own curiosity; indispensable to their success were the many public, searchable, large-scale genomic datasets becoming available at the time. It is a reminder to us all that basic, curiosity-driven research lies at the foundation of most of science’s success stories; that the aggregation of “big data” yields unexpected insights; and that even seemingly-insignificant parts of the planet’s biodiversity will reward our attention and understanding.
By Robin Floyd