Broad researchers describe new CRISPR-based method called Prime Editing

The goup of David Liu at the Broad Institute has published a paper in Nature describing a novel approach to edit genomes. The method, called "prime editing" relies on a fusion between a catalytically impaired Cas9 enzyme and a reverse transcriptase. Prime editing directly writes genetic information into the genome using just a single guide RNA. No double strand breaks or exogenous DNA templates are needed for the modification.

Since it was unveiled in 2012, CRISPR-Cas9 based gene editing has become the de facto standard for introducing genetic changes into the genomes of a wide array of species, from yeast to mammals. The basic mechanism of CRISPR-Cas9 employs a guide DNA to target the enzyme to a defined location within the host genome, where the Cas9 enzyme effects a double strand cut in the DNA. Subsequent repair by the NHEJ pathway results in insertions and deletions (indels) which may cause a frameshift and consequent inactivation of the target gene. Alternatively, exogenous pieces of template DNA can be introduced, resulting in deletions (knock-outs) or insertions (knock-ins) within the target gene.

While CRISPR-based approaches have been used extensively in the years since its first publication, the system is stil plagued by a number of shortcomings, the lack of specificity (off-target editing) and a lack of efficiency in altering larger tracts of DNA among them.

Prime editing, a method presented by researchers from the Broad Institute, promises to overcome some of these hurdles. The method allows accurate insertion or deletion of DNA, as well as being able to introduce single base pair changes into the genomic DNA. David Liu likens prime editors to word processors, capable of searching for target DNA sequences and precisely replacing them. They thus offer more targeting flexibility and greater editing precision than the original CRIPSR-Cas9 systems. Prime editing uses just one piece of guide RNA, which contains both the targeting sequence and the sequence which is supposed to be inserted into the genome. A fusion protein - part Cas9, part reverse transcriptase - then copies the relevant edits over into the genomic DNA.

Thus far, Liu's group has carried out more than 175 genome edits, including deletions, insertions and all types of base pair changes. They were also able to correct the gene defects underlying two diseases - sickle cell anemia and Tay-Sachs disease - by directly editing the genome of human cells with high efficiency and causing minimal off-target effects.

Clearly, more research is needed to explore the system further and to understand its capabilities and limitations, but as David Liu points out, prime editing has the potential to correct about 89% of all known pathogenic human genetic variants.

Anzalone et al. (2019) Nature: Search-and-replace genome editing without double-strand breaks or donor DNA