New – you know CRISPR, now it’s Prime Editing

This month, a new leap forward in gene editing has been published in Nature under the title ‘Search-and-replace genome editing without double-strand breaks or donor DNA’. DOI: 10.1038/s41586-019-1711-4. Let’s take a look at prime editing to get a clear overview.

Features of prime editing

12 possible mutations

prime editing mutations
The 12 possible mutations compatible with prime editing

Editing from 1 to 44 bases

Prime editing allows point change to maximum 44-nt long knock-in and maximum 80-nt long knock-out.

Though tagging of fluorescence is not possible, the prime editing platform can add a flag tag and a 6-histine tag that would be useful to isolate endogenous protein in native complexes. It is also a solution when an antibody is not available to locate the protein into the cells.

We can also note that the CRE-Lox system requires only 34 nt with 2 recognition regions of 13bp and 1 spacer region of 8bp. Thus, larger insertion can be performed with a successive combination of the prime editing and the CRE-Lox system using CRE mRNA.

KO of up to 26 codon is also possible with prime editing platform.

Knock-in with no donor

CRISPR-CAS9 gene editing leads to KI using a donor template to repair the double stranded break cause by the endonuclease activity of the CAS9. Without a donor, the classic CRISPR-CAS9 system leads only to KO and so lost of function.

Prime editing provides means to generate changes from 1 up to 44 bases without a donor. Thus, a transient expression of the prime editing complex is enough. There is no risk of genotoxicity caused by random insertion of a plasmid donor and the delivery into the cells is simpler and so, it should more efficient. These 2 key points provide interesting therapeutics perspectives for the up to 75000 genetic diseases.

How does the prime editing platform work?

Andrew Anzalone et al use the Cas9 without endonuclease activity with fusion to a reverse transcriptase enzyme. That “RT-dCAS9” is combined with a prime editing guide RNA (pegRNA). That short RNA is designed to target the specific site as per the classic CRISPR-CAS9. The difference is that it is extended to be reverse transcribed by the RT activity providing single stranded DNA to the targeted site. Then the edited DNA replaces the original DNA at the targeted site thanks to the repair machinery of the cells.

To an extent, we can say that the donor template is produced into the cells by reverse transcription from the pegRNA.
Prime editing is more efficient with less off-target effect than the classic CRISPR-Cas9. It is a safer way to make gene editing and to correct genetic-based diseases.

Further reading

You might be interested in this article on GEN Genetic Engineering and Biotechnology News: Genome Editing Heads to Primetime

Written by Dimitri Szymczak, PhD
Dimitri Szymczak is a Technical Support Specialist and Product Manager at tebu-bio, and a fan of capoeira in his spare time.