Enhancing prime editing efficiency and flexibility with tethered and split pegRNAs

Prime editing (PE) has advantages for small insertion, deletion or point mutations without double-stranded DNA breaks. The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity. Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA, which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs). sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells. We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.

. In addition, pegRNA circularization might also result in self-inhibition and compromise the PE efficiency (Liu et al., 2021). We have shown that the dynamics of CRISPR DNA targeting limits genome editing efficiency (Ma et al., 2016).
Here we used CRISPR-based genome imaging (Ma et al., 2016(Ma et al., , 2018 to compare the target efficiency of CRISPR-based GE (Genome Editor) and PE (Prime Editor). Fluorescent Cas9-sgRNA complex effectively targeted to chromosome 3-specific tandem repeats (C3) allows to be visualized under microscopy in U2OS cells (Fig. S1) (Ma et al., 2016). As shown in Fig. S1B and S1C, 2-4 bright foci were observed in the GE system but not the PE system suggesting that 3ʹ-terminal RTT-PBS of pegRNA resulted in low target efficiency of PE. We added the stem-loop aptamer MS2 (Convery et al., 1998) at the 3ʹ-terminal of pegRNAs (pegRNA-MS2) and found that visualization of C3 loci was recovered ( Fig. S1B and (S1C). We assume that the 3ʹ-terminal pegRNA tethered to Cas9 nickase will stabilize the PE-pegRNA complex. We fused tandem MS2 coat protein (tdMCP) to the N-terminal of Cas9 for binding 3ʹ-terminal MS2 at the engineered pegRNA. As we can see in Fig. S1B and S1C, C3 labeling was maintained. The low targeting efficiency of canonical PE suggests that inefficient targeting of genomic loci may compromise the PE efficiency. On the contrary, the recovery of C3 loci visualization in 3ʹ-terminal MS2 tagged pegRNA or tethered to Cas9 nickase indicates the engineered pegRNA or tethered to Cas9 nickase may improve the PE efficiency.
PE efficiency varies in different cell types (Chen et al., 2021;Nelson et al., 2022). To ensure that the improvement in PE efficiency by sPEs or tPEs was not limited to HEK293FT cells, we tested the above 10 loci with sPE-MS2 or tPE-MS2 in U2OS ( Fig. S2A and S2B) and HeLa cells using PE3 (Fig. S2C and S2D). In either U2OS or HeLa, sPE-MS2 or tPE-MS2 resulted in improvements in editing efficiency compared to canonical PE, averaging 2.5-or 3.1-fold higher editing in U2OS cells (Fig. 1H) and 3.7-or 2.7-fold higher editing in HeLa cells (Fig. 1I), with no significant change in edit/ indel ratios overall ( Fig. S3A and S3D). These results indicate that sPE and tPE can enhance PE efficiency in different cell types. We examined off-target editing by sPE-MS2 and tPE-MS2 for DYRK1A, RUNX1, BCL11A, SRD5A3, and JAK2 loci in HEK293FT cells. The off-target sites were predicted by Cas-OFFinder (Bae et al., 2014). Average <0.1% off-target prime editing was detected in canonical PE, sPE-MS2, and tPE-MS2 at the predicted off-target sites for each protospacer of DYRK1A, RUNX1, BCL11A, SRD5A3, or JAK2 in HEK293FT cells (Fig. S6).
The prime editing has the advantages for point mutations, small deletions, and insertions. However, the instability or misfolding of pegRNAs may have limited its applications for direct insertion of bigger size fragment such as >100 nucleotides. It will be interesting to test whether sPEs or tPEs will allow for the installation of DNA fragments with hundreds of nucleotides. There are several dual pegRNA strategies to increase the efficiency and precision of small or large deletions, small fragment insertions (Anzalone et al., 2022;Choi et al., 2022). Large fragment insertion has also been achieved by the combination of dual-pegRNA mediated small insertions and recombinase-mediated site-specific genomic integration (Anzalone et al., 2022). It will be intriguing to test whether sPEs or tPEs will benefit these dual-pegRNA systems since sPEs and tPEs showed better targeting efficiency than canonical PEs.
Tethered PEs offer the opportunity to liberate the RTT-PBS unit from the pegRNAs and spatiotemporally control the PEs. pegRNA in tPEs was separated to be conventional sgRNA and prime RNA containing PBS, RTT, and RNA aptamer resulting in SnPEs. One of the potential applications of SnPEs is more readily prepared by chemical synthesis of split pegRNAs due to the smaller sizes of sgRNA and pRNA. Synthetically modified sgRNA and prime RNAs may further enhance the PE efficiency. The SnPEs could also combine with inhibitors of DNA mismatch repair (MMR) (Chen et al., 2021) to further increase prime editing efficiency and precision. Separated prime RNA could be also introduced under the control of chemicals or lights (Stanton et al., 2018) and evolve the PE system to be tunable in space and time.