Enhancement of prime editing via xrRNA motif-joined pegRNA

Abstract

The prime editors (PEs) have shown great promise for precise genome modification. However, their suboptimal efficiencies present a significant technical challenge. Here, by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3′-extended portion of pegRNAs for their increased resistance against degradation, we develop an upgraded PE platform (xrPE) with substantially enhanced editing efficiencies in multiple cell lines. A pan-target average enhancement of up to 3.1-, 4.5- and 2.5-fold in given cell types is observed for base conversions, small deletions, and small insertions, respectively. Additionally, xrPE exhibits comparable edit:indel ratios and similarly minimal off-target editing as the canonical PE3. Of note, parallel comparison of xrPE to the most recently developed epegRNA-based PE system shows their largely equivalent editing performances. Our study establishes a highly adaptable platform of improved PE that shall have broad implications.

Prime editing for precise and highly versatile genome manipulation

“…3a, Table 1). In similar independent studies, other groups have added a Zikavirus exoribonuclease-resistant RNA motif (xr-pegRNA), a G-quadruplex (G-PE), or a stem-loop aptamer (sPE) to the pegRNA 3ʹ extension and have shown comparable improvements to prime editing in mammalian cell culture [74][75][76] (Fig. 3a, Table 1).…”

“…In their enhanced prime editing system (ePE), these extended pegRNAs are also fused to a nicking sgRNA and co-delivered with Csy4 nuclease, 70 , pegRNAs carrying the F + E crRNA scaffold 54,78 , and ePE pegRNAs 77 improve prime editor and pegRNA expression. epegRNAs 54 , ePE pegRNAs 77 , xr-pegRNAs 74 , G-PE pegRNAs 75 and sPE pegRNAs 76 reduce pegRNA degradation. PE2* 68 , PEmax 53 , CMP-PE-V1 63 and hyPE2 71 improve localization and/or DNA targeting of the prime editor complex.…”

Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders

“…However, PE-mediated correction of pathologic phenotype has been demonstrated ( Zhang H. et al, 2022a ; Zhang H. et al, 2022b ). Different strategies to improve PEs have been developed, either stabilizing the pegRNA ( Liu et al, 2021 ; Zhang G. et al, 2022 ), or manipulating repair pathways ( Chen et al, 2021 ; Ferreira da Silva et al, 2022 ), or a combination of both ( Chen et al, 2021 ; Jiang and Yao, 2022 ). Furthermore, by inserting specific same-sense mutations in the reverse transcription template of pegRNA or by altering the pegRNA secondary structure, efficiency could be significantly improved ( Li et al, 2022 ).…”

Prime Editing: An All-Rounder for Genome Editing

“…They also adopted the second strategy: introducing same-sense mutations at appropriate positions in the RT template portion of pegRNA (spegRNA), which increased the base-editing efficiency by an average of 353-fold. Zhang et al resisted pegRNA degradation by appending a viral exoribonuclease-resistant RNA motif (xrRNA) to the 3′ end extension of pegRNA, and the resulting xrPE had 3.1-, 2.5-, and 4.5-fold enhancements, respectively, for base conversions, small insertions, and small deletions in a variety of cell lines compared to PE3 [ 32 ]. Recent studies have also reported the modification of pegRNA with a human telomerase RNA (hTR) G-quadruplex (G 3 N 1-3 G 3 N 1-3 G 3 N 1-3 G 3 ) to improve the stability of pegRNA, resulting in G-PE with editing efficiency similar to that of the PE system using evopreQ1, while the G-quadruplex is shorter than evopreQ1 and xrRNA [ 33 ].…”