WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing

Abstract

Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3′ extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3′ terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.

Prime editing for precise and highly versatile genome manipulation

“…2), using two pegRNAs to simultaneously edit both DNA strands expands the size and type of genomic manipulations possible with prime editing. Many prime editing approaches that use two pegRNAs have been reported, including dual-pegRNA, HOPE (homologous 3′ extension mediated prime editor), PRIME-Del, PEDAR (PE-Cas9-based deletion and repair), twinPE, GRAND (genome editing by RTTs partially aligned to each other but non-homologous to target sequences within duo pegRNA), Bi-PE, bi-WT-PE and PETI (prime editor nuclease-mediated translocation and inversion) [55][56][57]99,[101][102][103][104][105] (Fig. 4a).…”

“…with an edited 3′ overhang that can be incorporated into the genome but also result in frequent formation of insertion or deletion (indel) byproducts 104,106,107 without designed homology to the genome. Importantly, because the newly synthesized 3ʹ flaps are typically dissimilar to the target site, they hybridize to each other with minimal competition from genomic sequence.…”

“…However, although PEDAR can make these desired editing outcomes with up to 27% efficiency, they are typically outnumbered by indel byproducts. In independent studies, Yao, Kim, and their respective co-workers developed bi-directional WT-PE (bi-WT-PE) and PETI systems that use a Cas9 nuclease prime editor similar to PEDAR 104,105 (Fig. 4a).…”

“…Creating a DSB instead of a DNA nick at the junction of flap synthesis may circumvent these steps and engage other DNA repair pathways to resolve the intermediate towards the intended edit, albeit with the undesired consequence of inducing more indel byproducts from endjoining processes. As discussed above, PEDAR, bi-WT-PE and PETI use this approach to mediate large sequence deletions, inversions and translocations 57,104,105 .…”

Marker-free co-selection for successive rounds of prime editing in human cells

“…This strategy yielded the PE4 (PE2 + MLH1dn), PE5 (PE3 + MLH1dn), and PE5b (PE3b + MLH1dn) editors 7 . In addition, nuclease prime editors have been shown to improve PE efficiency, but it comes at the expense of product purity 10 – 12 . Thus, the optimal PE strategy to adopt varies according to the context and there is a need to develop methods to consistently improve its success rate.…”