Engineered pegRNAs improve prime editing efficiency

Abstract

Prime editing enables the installation of virtually any combination of point mutations, small insertions, or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we demonstrate that degradation of the 3′ region of the pegRNA that contains the reverse transcriptase template and the primer-binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3 to 4-fold in HeLa, U2OS, and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3′ structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3′ motifs. Finally, we demonstrated that epegRNAs enhance the efficiency of the installation or correction disease-relevant mutations.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

“…In vitro transcription of prime editor and MLH1dn mRNA used in iPSC and T cell experiments As described previously (Nelson et al, 2021), plasmids were cloned to encode an inactivated T7 promoter followed by a 5 0 untranslated region (UTR), Kozak sequence, coding sequences of PE2 or MLH1dn, and a 3 0 UTR. T7 promoter inactivation prevents potential transcription from circular plasmid template during mRNA generation.…”

“…Strategic installation of additional silent mutations nearby an intended edit can also improve prime editing efficiency by evading MMR recognition, even in the absence of MLH1dn. Finally, we engineered an optimized “PEmax” prime editor architecture that further increased editing efficiency in synergy with PE4, PE5, and engineered pegRNAs (epegRNAs) ( Nelson et al, 2021 ). These findings deepen our understanding of prime editing and establish prime editing systems with substantially improved efficiency and outcome purity across 191 different edits at 20 loci in seven mammalian cell types.…”

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

“…To determine if coselection could streamline the production of homozygous cell lines for functional studies, we designed pegRNAs to install well-characterized and clinically relevant MTOR hyperactivating mutations 49,50,52,53 or rapamycin resistance mutations [54][55][56] . While the design and screening of several pegRNAs remains a critical aspect of successful PE 1,7,12 , we designed and tested only one pegRNA per target based on previously established rules 1,5 for this proof-of-concept experiment.…”

“…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.To circumvent these limitations several approaches have been implemented including editing with purified ribonucleoprotein complexes (RNPs) 10 , mRNA-based delivery 7,11 , engineered pegRNAs (epegRNAs) 12 , NLS-and codon-optimized prime editors 7,9,13 , enrichment with puromycin, and fluorescent reporter-based selection [14][15][16] . Nevertheless, these improvements are still dependent on extensive pegRNA optimization, absolute activity remains low, and the streamlined production of homozygous cell lines has yet to be achieved 1,12 . Hence, further refinements to these approaches are needed to boost the efficiency of PE in human cells 2,3,7,12 .We previously reported a robust coselection method for CRISPR nucleases that relies on cotargeting a gene of interest (GOI) and the ATP1A1 locus to confer dominant cellular resistance to ouabain 17 , a plant-derived inhibitor of the ubiquitous and essential Na + /K + ATPase 18,19 .…”

“…Nevertheless, these improvements are still dependent on extensive pegRNA optimization, absolute activity remains low, and the streamlined production of homozygous cell lines has yet to be achieved 1,12 . Hence, further refinements to these approaches are needed to boost the efficiency of PE in human cells 2,3,7,12 .We previously reported a robust coselection method for CRISPR nucleases that relies on cotargeting a gene of interest (GOI) and the ATP1A1 locus to confer dominant cellular resistance to ouabain 17 , a plant-derived inhibitor of the ubiquitous and essential Na + /K + ATPase 18,19 . Such an approach derives from the observation that simultaneous targeting of two different loci results in double-editing events that are not statistically independent 17,[20][21][22][23][24][25] .…”