The huge publicity given recently to genetic manipulation has meant that almost everyone has heard of CRISPR-Cas — it’s rapidly become the most popular way of editing genes from any organism. Guide RNA is designed to bind to a specific DNA sequence whereupon the recruitment of molecular scissors (the Cas9 enzyme) cuts the DNA at that sequence. The cell’s repair mechanisms will either glue the cuts back together or insert a novel stretch of DNA if that is delivered to the cell — we outlined the basic idea in Re-writing the Manual of Life.
There’s no doubt that, versatile and precise, the development of CRISPR in the last decade or so has been one of the great advances in the life sciences and in medicine it has led to ‘designer’ immune cells with enhanced abilities to seek and attack tumours, as we described in Gosh! Wonderful GOSH.
Seeing a New World described a rat model of retinitis pigmentosa, a genetic disease that is a major cause of inherited blindness and afflicts about one and a half million people worldwide (one in 4,000 in the UK). In this model it’s now possible to inject under the retina of rats with the disease an inert virus carrying the bits of CRISPR-Cas plus a replacement gene for the one damaged in retinitis pigmentosa. The eyesight of these treated mice recovered substantially in response to CRISPR.
Almighty Power showed how CRISPR could make almost 4,000 different versions (variants) of the BRCA1 protein and Shifting the Genetic Furniture described using the method to move DNA around within the nucleus.
Wonderful though all this is, CRISPR is not without its problems, most notably that it is not 100% efficient, there can be off-target effects and, as might be predicted, the act of cutting DNA activates TP53 which can be toxic to the cell (Caveat Emptor).
CRISPR base editing
The most recent advance in genome editing uses parts of CRISPR together with other enzymes to insert point mutations into cellular DNA or RNA directly without making double-stranded DNA breaks. The method still uses guide RNAs for targeting but adds a second enzyme to Cas9. Depending on the enzyme either cytidine is converted to thymidine or adenosine to guanosine (A to G mutation).
Base editing by CRISPR. The CRISPR system is modified by coupling another enzyme to Cas9 (or to ‘dead’ Cas9 — dCas9). The enzyme shown is cytidine deaminase. The PAM site (protospacer adjacent motif) is a short DNA sequence following the cleavage site and is required for a Cas nuclease to cut. Alternatively, an E.coli enzyme can be used to make an adenine base editor. From https://www.addgene.org/crispr/base-edit/
Treating human disease by CRISPR
Leber Congenital Amaurosis (LCA) is a spectrum of inherited conditions that cause poor vision due to a defect in the cells that detect light in the retina (rods and cones). A press release on October 10, 2019 described LCA patients treated with sepofarsen (QR-110) experiencing a rapid and durable improvement in vision. Sepofarsen uses chemically modified nucleotides complementary to specific mRNAs in the cell (it’s an ‘antisense’ therapy). Sepofarsen tackles a specific mutation in the CEP20 gene by repairing the mRNA, hence permitting a normal CEP20 protein to be made. The drug is administered by injections into the vitreous of the eye (intra-vitreal injections).
A further step in using direct administration of CRISPR–Cas9 gene therapy into the body to treat LCA has come in the form of a trial named BRILLIANCE. Two pharmaceutical companies, Editas and Allergan, are co-operating in this endeavour using EDIT-101, a CRISPR-based gene-editing treatment delivered by an adeno-associated virus, AGN-151587. As with sepofarsen, the components of the gene-editing system are injected directly into the eye, near photoreceptor cells. The first patient has been treated in a phase 1/2 trial of AGN-151587, receiving a single subretinal injection of AGN-151587 and details of the ongoing trial can be found on www.clinicaltrials.gov.
These early trials are not, of course, ‘fixing’ cancer but they do appear to give a ‘proof of principle’ that it should be possible to use gene editing to re-activate, for example, mutated tumour suppressors. Watch this space!!