Gene editing is the hope for curing genetic diseases. The prime editing (PE) technology developed by Broad Institute David Liu's group and others in 2019 can use RNA as a template to perform various editing such as deletion and replacement without introducing DNA double-strand breaks, and has good clinical prospects. However, this technology has the problem of unstable gene editing efficiency in different cell lines at different genomic sites (1, 2).
prime editing gene editing technology (1)
In order to further understand the mechanism of prime editing gene editing technology and make it efficient and stable. David Liu's group collaborated with Princeton University Britt Adamson's group and others to screen intracellular factors that affect the efficiency of prime editing through CRISPRi screening and other technologies (2).
CRISPRi screens factors that affect prime editing efficiency in cells (2)
After screening, it was found that DNA mismatch repair pathway (DNA mismatch repair (MMR)) , which is a key molecule that inhibits DNA mismatch repair, affects the efficiency of prime editing. MLH1, or designing several more synonymous mutations in the template RNA to escape DNA mismatch repair, can improve the efficiency of prime editing, and in some cases (a PE3 version that adds gaps in the non-editing strand to improve the efficiency of prime editing) improves the purity of gene editing (the ratio between target edits and irrelevant indels increases) .
Key factors of the DNA mismatch repair pathway affect the efficiency of prime editing (2)
The researchers further combined the optimized fusion protein (PEmax; adjusted through reverse transcriptase codon optimization, linker and nuclear localization sequence redesign, Cas9 protein mutation, etc. ) and template RNA (engineered pegRNAs (epegRNAs), adjust the 3' end sequence) have constructed the best prime editing gene editing system currently.
inhibits DNA mismatch repair and cooperates with PEmax and epegRNAs to achieve optimal prime editing effects (2)
Research believes that this work expands the understanding of the gene editing mechanism of prime editing and improves its usability.
This work was published in Cell(2) on October 14.
Comments:
A very practical work; however, it should be noted that CRISPRi screens and edits exogenous sites in the article, and the efficiency of exogenous prime editing may be different from the factors affecting the editing efficiency of endogenous DNA prime editing in cells; follow-up Screening of factors affecting the efficiency of prime editing of endogenous DNA sequences can further provide valuable information.
In addition, whether this optimization method is suitable for non-dividing cells is also an issue that requires further research.
Also, a very clear and noteworthy data in the article is that the position of the "gap" in the non-editing chain has a great impact on prime editing. Subsequently, more detailed adjustment of this "gap" is also an important optimization direction; however, there are The problem is that the optimal "gap" setting is affected by sequence, chromatin status, etc. In the future, more systematic research on chromatin status and prime editing efficiency is needed.
The "gap" position of the non-editing chain seriously affects the editing effect of prime editing (2)
Corresponding author's introduction:
https://www.hhmi.org/scientists/david-r-liuht3
References:
. A. V. Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature.576, 149–157 (2019).
. P. J. Chen et al., Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 0 (2021), doi:10.1016/J.CELL.2021.09.018.
Original link:
https://www.cell.com/cell/fulltext/S0092-8674(21)01065-5
