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 show 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 3-4-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 showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.

Figure 1.. Truncated pegRNAs limit prime editing efficiency.

(a) (left) Schematic of a prime editing complex composed of a prime editor (PE) protein that consists of a Cas9 nickase (nCas9) fused to a modified reverse transcriptase via a flexible linker and a prime editing guide RNA (pegRNA). (right) Degradation of the 3′ extension of a pegRNA by exonucleases could impede editing efficiency through loss of the PBS. (b) PE3-mediated editing efficiencies with the addition of plasmids expressing sgRNAs, truncated pegRNAs that target the same genomic locus (HEK3), non-targeting pegRNA, or SaCas9 pegRNAs. All pegRNAs are expressed from a U6 promoter. Data and error bars reflect the mean and standard deviation of three independent biological replicates. (c) Design of engineered pegRNAs (epegRNAs) that contain a structured RNA pseudoknot, which protects the 3′ extension from degradation by exonucleases.

Figure 2.. PE editing efficiency is enhanced by the addition of structured RNA motifs to the 3′ terminus of pegRNAs.

(a) Efficiency of PE3-mediated insertions of the FLAG epitope tag at the +1 editing position (insertion directly at the pegRNA-induced nick site) across multiple genomic loci in HEK293T cells using canonical pegRNAs (“unmodified”), pegRNAs with either evopreQ₁ or mpknot appended to the 3′ end of the PBS via an 8-nt linker, or pegRNAs appended with only the 8-nt linker sequence. (b) Summary of the fold-change in PE editing efficiency relative to canonical pegRNAs of the indicated edit at various genomic loci upon addition of the indicated 3′ motif via an 8-nt linker, or the addition of the linker alone. “Transversion” denotes mutation of the +5 G•C to T•A at RUNX1, EMX1, VEGFA, and DNMT1, the +1 C•G to T•A at RNF2, and the +1 T•A to A•T at HEK3, where the positive integer indicates the distance from the Cas9 nick site. “Deletion” denotes a 15-bp deletion at the Cas9 nick site. Data summarized here are presented in (c) and Supplementary Fig. 2. The horizontal bars show the median values. (c) Representative improvements in PE editing efficiency from appending either evopreQ₁ (p) or mpknot (m) via an 8-nt linker to pegRNAs with varying template lengths (in nucleotides, indicated). (d) Editing activities of canonical pegRNAs and modified pegRNAs across three genomic loci in HeLa cells, U2OS cells, and K562 cells. Data and error bars in a, c, and d reflect the mean and standard deviation of three independent biological replicates.

Figure 3.. Structural motifs increase RNA stability and efficiency of reverse transcription but reduce Cas9 binding affinity.

(a) Resistance of unmodified pegRNA or epegRNA containing evopreQ₁ or mpknot to degradation upon exposure to HEK293T nuclear lysates. The agarose gel shown is representative of three experiments. Untreated in vitro transcribed pegRNAs or epegRNAs served as standards. Percent RNA remaining was calculated using densitometry. Significance was analyzed using a two-tailed unpaired Student’s t test (p=0.0028 for mpknot and 0.0022 for evopreQ₁). (b) Fold change in abundance of the pegRNA scaffold relative to unmodified pegRNA upon exposure to HEK293T nuclear lysates in the absence and presence of nCas9 as determined by RT-qPCR of the sgRNA scaffold. (c) Comparison of prime-editing intermediates generated by PE2 with either pegRNAs or epegRNAs at RNF2. Dotted lines indicate the full-length reverse transcriptase product templated by the pegRNA or epegRNA tested at the indicated locus. X axis is relative to the position of the PE2-induced nick with the first base 3’ downstream represented as position +1. Histograms and pie charts are generated from the average of three independent biological replicates. (d) PE3 editing efficiencies in HEK293T cells using unmodified pegRNAs, pegRNAs containing the evopreQ1 motif, or pegRNAs containing a G15C point mutant of evopreQ₁ (M1) that disrupts pseudoknot motif structure. (e) Fraction of Cas9 RNPs composed of dCas9 and either unmodified pegRNA or epegRNA containing either evopreQ₁ or mpknot and templating a +1 FLAG tag insertion at HEK3 bound to dsDNA as determined by MST. (f) CRISPRa transcriptional activation by pegRNAs, epegRNAs, and sgRNAs. Reported GFP fluorescence is normalized to iRFP fluorescence expressed from a co-transfected plasmid. AU, arbitrary units. (g) Fraction of unmodified pegRNA or epegRNA (templating a +1 FLAG tag insertion at HEK3) containing either evopreQ₁ or mpknot bound to H840A nCas9 as determined by microscale thermophoresis (MST). Data and error bars reflect the mean and standard deviation of three independent biological replicates.

Figure 4.. Prime editing-mediated editing efficiency of therapeutically relevant genome editing is improved by the use of epegRNAs.

(a) PE3-mediated installation of the G127V mutation in PRNP that protects against human prion disease^,, and (b) correction of the pathogenic c1278TATC insertion in HEXA that causes Tay Sachs disease in both HEK293T cells and (c) primary patient-derived fibroblasts. (d) Comparison of PE2-mediated installation of pathogenic and protective alleles using unoptimized epegRNAs or unoptimized pegRNAs at nine genomic sites. Reference SNP (rs) designations can be found for all mutations in Supplementary Table 6. Data and error bars in a, d, and e reflect the mean and standard deviation of three independent biological replicates.