Researchers from the Liu Group at MIT have developed a new gene editing tool named prime editing using a novel strategy. This approach is less prone to incorrect edits than traditional Cas-9 editing couple to homologous DNA recombination (HDR) [EE1] . The researchers claim that this editing technique could correct about 89% of known pathogenic human genetic variants.
The structure of the prime editor consists in a fusion protein that comprise a reverse transcriptase (RT) fused to a modified Cas 9 enzyme able to cut only in one of the strands of double stranded DNA template. In addition, the editing system contains an RNA sequence that can work as a template for the RT followed by a traditional RNA guide sequence that is used to target the fusion protein to the DNA sequence region to be edited. This optimized guide RNA named pegRNA contains the new genetic information that will replace the target. Targeting the fusion protein to the pegRNA bound to the target DNA will cut one of the DNA strands in the proximity of the nucleotides to be corrected. The resulting free 3’ hydroxyl group of the DNA, primes the reverse transcription of the edited gene using part of the pegRNA as a template. The result of this process leaves behind an intermediate that has two redundant flaps, the 3’ flap contains the edit while the 5’ flap is unedited. The 5’ flap is a preferred substrate for endonucleases and is removed, allowing the edited flap to integrate into the chain.
So, now we have a DNA molecule with the edited sequence in one strand and the original sequence in the other strand. The next step would be that the Cas9 protein with a proper guide RNA targets the original strand in the canonical described way and cleaves it. This action triggers the DNA reparation system of the cell to solve this problem by using the modified strand (with the interested gene) as a template. Finally, we obtain the double strand DNA molecule with the interested gene in a specific site.
This new mechanism differs from CRISPR-Cas9 mainly by:
- The Cas9 protein has been modified in order to only cut one strand of the DNA molecule instead of cutting two strands and leaving the two ends free.
- The reverse transcriptase, an enzyme that can generates a DNA sequence from a RNA template sequence.
- pegRNA (prime editing guide RNA), which has two main function: to guide the cas9 protein in a specific place of the DNA; and also acts as a template for the synthesis of DNA by the reverse transcriptase. However, in CRISPR-Cas9 it is need the DNA as a template to introduce a change in the genome.
So, basically the prime editing is a more precise technique and with less mistakes due to there is less probability of mutations because while the pegRNA is binding the DNA, the Cas9 is cutting the strand and the reverse transcriptase is synthesizing the new DNA strand all at the same time, so there is less probability of being mutated.
In CRISPR-Cas9, is limited because it can make changes in the genome while prime editing apart from change completed sequences it can also change particular nucleotides; also, CRISPR-Cas9 is based on using the cell reparation system that introduce random mutations, while in prime editing changes are based on pegRNA.
However, it also has limitation this new technique because it does not allow big insertions neither deletions due to the changes that are being produced in the genome are in the pegRNA and the size it must be limited in order to do not get degraded or eliminated by the cell. Unspecific or undesired DNA modification cannot be completely excluded either.
To conclude briefly, in our opinion we think that CRISPR-Cas will not disappear because is a better technique to inactivate genes as it is not necessary to be extremely specific and it is more easy than prime editing that requires specificity. So, for repairing or inserting new genes we think that would be better prime editing but for inactivating genes it can be also use CRISPR-Cas technique with the same efficacy.
Vicent Tur and Fernando Gómez.
2º Grade of Biotechnology. Microbiology Course. Faculty of Pharmacy. Universidad CEU San Pablo.
[EE1]
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