By Angela Lek, PhD
The discovery of CRISPR-Cas9 gene-editing technology has undoubtedly revolutionized the field of human genetics, enabling for the first time in human history the ability to target disease-causing mutations in our DNA–the root cause of genetic diseases. Now a new, improved version of CRISPR-Cas9 has been developed that extends the capabilities, as well as fixes some problems uncovered in the original version.
Correcting disease-causing mutations using classic CRISPR-Cas9 technology requires a molecule called Cas9 (originally found in bacteria) to act as “molecular scissors” to cut the DNA molecule. Cas9 can be targeted to a specific DNA mutation and then a correct (non-mutated) stretch of DNA can be put in its place. Think of it as a “cut and paste” method to fix DNA and faulty genes.
One drawback of the technology is that a significant amount of unwanted DNA changes can also occur at the Cas9 cut site. Imagine that your spell check not only removes the misspelled word but introduces new errors – not good! Six years following the publication of this technology, scientists are concerned with the unpredictability of classic CRISPR-Cas9 technology as a strategy for correcting genetic mutations in humans.
A recent study published by the Liu lab at Broad Institute in Cambridge, Massachusetts, reports on a new type of CRISPR-Cas9 technology referred to as “prime editing” that allows for targeted insertions, deletions, and all possible base conversions without some of the problems seen in the original technology and with increased efficiency. This new transformative technology utilizes a single sequence that specifies both the DNA target and the new genetic information for replacement, thus nick-named ‘search and replace’ genome editing.
Importantly, the authors report that the new version results in low unintended effects (“off-target activity”) and overcomes many of the limitations imposed by classic CRISPR technologies, thus making it a more attractive tool for correcting human disease mutations.
Prime editing has been applied to human cells in culture to reverse genetic mutations underlying sickle cell disease and Tay-Sachs disease. For FSHD, prime editing technology has the potential to target and disable the poly-adenylation signal associated with the 4qA (“disease permissive) haplotypes in patients – one of the key disease-enabling signals associated with FSHD.
It is important to note that correcting mutations in patient cells in a laboratory dish does not easily translate into doing the same in actual patients. Although the technology now exists to reverse targeted mutations, we are still faced with the challenge of delivering it to disease-affected cells in the human body. The most promising mode of delivery that has been demonstrated for CRISPR technology for targeting to muscle cells in an animal model is using AAV (adeno-associated virus) to shuttle CRISPR-Cas9 into the cell’s nucleus (where the DNA is). In its current state, the prime editing system is too big to fit into AAV and will require additional modifications to so it can work with AAV.
We are living through a golden age for human genetics research with the development of technologies to read and make precise changes to DNA. However, in order to realize the full potential of these technologies, we need a means of safely and efficiently deliver them to affected cells in the human body.
Many labs and companies around the world are currently dedicated to improving existing viral-delivery vehicles as well as devising novel nanoparticles for gene delivery.
Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Oct 21. doi: 10.1038/s41586-019-1711-4. [Epub ahead of print]