The discovery of CRISPR five years ago has revolutionised the way we can manipulate and edit DNA. The use of the technology in clinical trials has exploded, primarily in China, where, as at June 2017, as many as 20 human trials were due to be under way. Last week two groups from MIT and Harvard, working together at the Broad Institute, published papers in Science and Nature respectively, which show further promising developments to this revolutionary technology.
The CRISPR technology uses a combination of guide RNA (gRNA) coupled with a bacterial nuclease, Cas9. The gRNA guides the Cas9 to a specific string of DNA bases in the relevant section of DNA and the nuclease cuts the DNA at a specific point in the string. The complex then detaches and the cellular repair mechanisms attempt to re-join the cut ends. As cellular repair mechanisms are inherently flawed, it often results in the addition or deletion of DNA bases, the effect of which (one hopes) will knock out the gene’s function. This technology can also be used to edit DNA by the insertion of strands of DNA which include altered DNA sequences, relying on homology-directed repair, another cellular repair mechanism. This technique, however, is inefficient, and is dependent on cellular division and so cannot function in non-dividing cells, such as those in the brain and muscle.
The Nature publication adapts this technique by providing an efficient and specific process for single base editing. The technique uses a dead Cas9 nuclease together with another enzyme, TadA. The dead Cas9 cannot cut the DNA, but merely unzips it and inserts a nick on one side of the helix. The TadA enzyme then converts adenosine (A), one of the four bases found in DNA, into inosine (I), a “foreign” base, at the relevant position. Cellular repair mechanisms then kick in, reading the “I” as a guanine (G), and so insert a cytosine (C)-G base pair, instead of an A-tyrosine (T) base pair. This has the potential to be used to correct disease-causing point mutations. Although a similar technique enabling C-G to T-A conversions has already been published, this publication has enabled programmable installation of all four base transitions.
The publication in Science takes CRISPR in a different direction by editing RNA instead of DNA. This technique is substantially similar to that used in the Nature publication, but uses a dead Cas13 together with ADAR, an enzyme similar to TadA. The gRNA is complementary to the bases on the cellular RNA except for the base which is intended to be changed. This causes the base to kick out from the helix and ADAR converts the A to an “I”. The cellular ribosomes, the protein-manufacturing enzymes, read this as a G, and so incorporate an alternative amino acid, altering the downstream protein.
RNA editing has opened up a new avenue into gene therapy without the need to alter a patient’s DNA. In addition to eliminating some of the ethical issues associated with DNA editing, the technique offers an opportunity for transient, and reversible, gene therapy as RNA is degraded in the cell. The researchers suggest this could be useful in “treating diseases caused by temporary changes in cell state”. For example, one could edit a protein when there are high levels of local inflammation, and switch off the process once the inflammation is gone.
The global gene editing market is predicted to reach $7.5bn by 2024, up from $518.5m in 2015 in the US alone. Investment into CRISPR-focussed biotechnology companies has soared, with Crispr Therapeutics going public with its $56m IPO on Nasdaq in Q4 2016, alongside Editas and Intellia earlier in that year. These further advances in this already ground-breaking technology should provide additional opportunities for biotechnology companies to take advantage of the wide-ranging application of these techniques in the clinic. We will be watching this space…