CRISPR Prime Editors Unleashed

Prime editing is known as the ‘search-and-replace’ cousin to the CRISPR/Cas genome editing technique.¹ Typically, prime editors are a Frankensteinian enzyme–a Cas nickase fused to a reverse transcriptase. This monstrous protein joins with a guide RNA called a prime editing guide RNA (pegRNA).

The prime editor ‘searches’ for the genomic target site using the 5′ end of the pegRNA and makes a single-strand cut with its nickase module. The resulting single-strand flap of genomic DNA binds to the 3′ end of the pegRNA, which serves as a primer and template for the reverse transcriptase module to proceed with ‘replacing.’ A secondary nick opposite the edited strand can be introduced by a nicking guide (ngRNA), which causes cellular DNA repair machinery to favor maintenance of the newly synthesized edited strand. Unlike traditional CRISPR/Cas genome-editing systems, prime editing does not rely on introducing double-strand breaks or ‘pasting’ of repair templates, which can result in a higher frequency of unintended mutations or ‘typos.’

While prime editing is a promising genome editing paradigm, delivery of prime editors has been challenging because of the size of the prime editor construct, especially for space-constrained delivery systems (e.g. AAV and lentivirus). In this SNiP, the authors “inadvertently discovered” that the reverse transcriptase module can function in trans w/ the Cas9 nickase module of the prime editor. The happy coincidence occurred when they were examining alternative prime editor architectures. While swapping fusion of the reverse transcriptase module from the N- to C- terminus of the prime editor, they found editing activity was not significantly different.

Because the two enzymes can be delivered separately without impacting editing activity, separate plasmids can be used to express the prime editing machinery. In addition to being a boon for space-constrained delivery systems, this also simplifies protein engineering efforts. For example, the authors were able to further improve on the reverse transcriptase module, including shrinking its size. Importantly, efficiency and off-target effects were not significantly different between using the intact prime editor and the split constructs, opening the door for further advancements of the prime editing technique for use in the development of gene and cell therapies.

Citation

Title: Engineered CRISPR prime editors with compact, untethered reverse transcriptases
Authors: Julian Grünewald, Bret R. Miller et al.
Journal: Nature Biotechnology, Volume 41, March 2023.
DOI: 10.1038/s41587-022-01473-1
Product Usage: Plasmid DNA mixtures (encoding intact or split prime editors, pegRNA and ngRNA) were transfected in HEK 293T cells in 24- and 96-well plates using TransIT-X2® Transfection Reagent. iCell® cardiomyocytes in 96-well plates were transfected with the plasmid DNA mixtures using TransIT®-LT1 Transfection Reagent.

Discover more ways TransIT® reagents were used for CRISPR research in the Mirus Bio Citations Database.