Modified pegRNAs mitigate scaffold-derived prime editing by-products

Abstract

Prime editors (PEs) employ reverse transcriptase (RT) to install genomic edits using a template within the prime editing guide RNA (pegRNA). RT creates a 3' genomic flap containing the intended edit. However, reverse transcription can continue beyond the template, incorporating the pegRNA scaffold sequence into the 3' flap. These scaffold-derived by-products can be installed alongside the intended edit, reducing prime editing precision. Here, we develop a method that prevents RT from accessing the scaffold, thereby mitigating such by-products. We demonstrate that an internal abasic spacer or 2'-O-methylation within the pegRNAs terminates RT at the end of the template. This prevents scaffold-derived sequences from being incorporated into the target locus. We benchmark these pegRNAs in different cell types and demonstrate that they can be used with processive PEs such as PE6d or PE**. Our findings provide a simple approach to mitigate a common prime editing by-product and improve prime editing precision.

Fig. 1. Abasic sites within synthetic pegRNAs precisely block reverse transcription by M-MLV RT.

a Schematic representation of pegRNA scaffold capture by NHEJ in the PRINS editing assay. PRINS editing results in unintended pegRNA scaffold integrations due to flap extension past the intended reverse transcription template (RTT). b PRINS editing using modified (M) pegRNAs to prevent RT from pegRNA scaffold readthrough. c Chemical structures of modifications within rSp-pegRNA and C3-pegRNA. C96 = cytosine at the position 96 of the SpCas9 pegRNA scaffold. d Scaffold incorporation analysis by amplicon-seq of PCSK9 editing in K562 cells electroporated with PEn mRNA and unmodified pegRNA, rSp-pegRNA, or C3-pegRNA. Plots show mean ± SD of n  =  3 biological replicates. e Representative CRISPResso2 alignment of PEn + pegRNA and PEn + rSp-pegRNA prime editing outcomes from experiment in Fig. 1d. Source data are provided as a Source Data file.

Fig. 2. PRINS editing with modified pegRNAs in human cell lines.

a PRINS editing at PCSK9, CTLA4, HBEGF and AAVS1 genomic loci in four different human cell lines (K562, HEK293T, HeLa and HepG2) using electroporation of PEn mRNA in combination with synthetic pegRNA, rSp-pegRNA or C3-pegRNA to install indicated small insertions (ins.). Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in the prime editing mode. Plots show mean ± SD of n  =  3 biological replicates. Prime edits precise = intended prime edits; Prime edits all (no scaffold) = precise prime edits + prime edits co-occurring with indels not derived from scaffold; Scaffold incorporated = prime edits with at least one additional nucleotide derived from scaffold; Indels = non-prime edited insertions or deletions. b Quantification of data from (a). “Precision score” was calculated as total number of amplicon-seq reads with precise prime edit per overall editing. “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. Error bars represent mean ± SD. Datapoints represent 4 different cell line experiments in (a). Statistical difference was determined using Student’s t test (paired, two-tailed). *P  <  0.05, **P  <  0.01, ***P  <  0.001. Calculated P values: PCSK9: pegRNA vs. rSp-pegRNA = 0.0032, pegRNA vs. C3-pegRNA = 0.0014; CTLA4: pegRNA vs. rSp-pegRNA = 0.0039, pegRNA vs. C3-pegRNA = 0.0057; HBEGF: pegRNA vs. rSp-pegRNA = 0.0034, AAVS1: pegRNA vs. rSp-pegRNA = 0.0154. Source data are provided as a Source Data file.

Fig. 3. PRINS editing with modified pegRNAs in primary human hepatocytes and mouse embryos.

a PRINS editing at PCSK9 and HBEGF genomic loci in primary human hepatocytes transfected with PEn mRNA and a synthetic pegRNA or rSp-pegRNA to install small insertions (ins.). Editing outcomes were analyzed by amplicon-seq and CRISPResso2 in the prime editing mode. “Precision score” was calculated as total number of amplicon-seq reads with precise prime edit per overall editing. “Prime edits with scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. Plots show mean ± SD of n  =  3 biological replicates. Statistical difference was determined using Student’s t test (paired, two-tailed). Prime edits precise = intended prime edits; Prime edits all (no scaffold) = precise prime edits + prime edits co-occurring with indels not derived from scaffold; Scaffold incorporated = prime edits with at least one additional nucleotide derived from scaffold; Indels = non-prime edited insertions or deletions; *P  <  0.05, **P  <  0.01, ***P  <  0.001. Calculated P values: PCSK9 “Prime edits - precise” = 0.005, PCSK9 “Prime edits with scaffold” = 0.0057, PCSK9 “Precision score” = 0.0104, HBEGF “Prime edits - precise” = 0.0123, HBEGF “Prime edits with scaffold” = 0.0018 HBEGF “Precision score” = 0.0027. b PRINS editing of mouse embryos using electroporation of PEn-pegRNA or PEn-rSp-pegRNA RNP complexes to install a 5 bp insertion into the endogenous Map3k15 locus. Each data point represents a single embryo edited with either pegRNA (n = 25) or rSp-pegRNA (n = 24). Single embryos were analyzed by amplicon-seq and CRISPResso2 after 5 days of in vitro cultivation. “Prime edits with scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. “Precision score” was calculated as total number of amplicon-seq reads with precise prime edit per overall editing. “Reads with precise edit” corresponds to a total number of precisely edited amplicon-seq reads in each embryo. Error bars represent mean ± SD. Statistical significance was determined using Student’s t test (unpaired, two-tailed). *P  <  0.05, **P  <  0.01, ***P  <  0.001. Calculated P values: “Prime edits with scaffold” <0.0001, “Precision score” = 0.011, “Reads with precise edit” = 0.0319. Source data are provided as a Source Data file.

Fig. 4. 2’-O-methylation of C96 in the pegRNA scaffold precisely blocks reverse transcription by M-MLV RT.

a Chemical structure of C96 methylation within the scaffold of C96Me-pegRNA. C96 = cytosine at the position 96 of the SpCas9 pegRNA scaffold. G95 = guanine at the position 95 of the SpCas9 pegRNA scaffold. RTT = reverse transcription template. b Scaffold incorporation analysis by amplicon-seq of PCSK9 PRINS editing in K562 cells electroporated with PEn mRNA and unmodified pegRNA or C96Me-pegRNA. c PRINS editing at PCSK9 and HBEGF genomic loci in four different human cell lines (K562, HEK293T, HeLa and HepG2) using electroporation of PEn mRNA and synthetic pegRNA or C96Me-pegRNA to install indicated small insertions (ins.). Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in the prime editing mode. Plots show mean ± SD of n  =  3 biological replicates. d Quantification of data from (c). “Precision score” was calculated as total number of amplicon-seq reads with precise prime edit per overall editing. “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. Error bars represent mean ± SD. Datapoints represent 4 different cell line experiments in (c). Statistical difference was determined using Student’s t test (paired, two-tailed). Prime edits precise = intended prime edits; Prime edits all (no scaffold) = precise prime edits + prime edits co-occurring with indels not derived from scaffold; Scaffold incorporated = prime edits with at least one additional nucleotide derived from scaffold; Indels = non-prime edited insertions or deletions. *P  <  0.05, **P  <  0.01, ***P  <  0.001. Calculated P values: PCSK9 precision score = 0.0016; PCSK9 prime edits containing scaffold = 0.0009; HBEGF precision score = 0.0052; HBEGF prime edits containing scaffold = 0.0054. e PRINS editing of Map3k15 locus in AML12 cells using lipofection of PEn mRNA and synthetic pegRNAs to install 5 bp insertion. Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in the prime editing mode. Plots show mean ± SD of n  =  6 biological replicates. Source data are provided as a Source Data file.

Fig. 5. Modified pegRNAs mitigate scaffold incorporation by PE3 and highly processive PE systems.

a Editing of FANCF with PE3 in K562 cells. Cells were electroporated with RNPs and either pegRNA or rSp-pegRNA to install indicated substitution at the FANCF genomic locus. b Quantification of data from (a). “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. c Editing with prime editing nucleases PEn6d and PEn** in HEK293T cells. Cells were transfected with plasmids expressing PEn6d or PEn** in combination with synthetic pegRNA or C96Me-pegRNA to install a 7 bp insertion (ins.) at the VEGFA genomic locus. d Quantification of data from (c). “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. e Editing with prime editing nickases PE6d and PE** in HEK293T cells. Cells were transfected with plasmids expressing PE6d or PE** in combination with synthetic pegRNA or C96Me-pegRNA to install a 7 bp insertion (ins.) at the VEGFA genomic locus. Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in the prime editing mode. f Quantification of data from (e). “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in the prime editing mode. Plots show mean ± SD of n  =  3 biological replicates. Prime edits precise = intended prime edits; Prime edits all = precise prime edits + prime edits co-occurring with indels; Scaffold incorporated = prime edits with at least one additional nucleotide matching scaffold; Indels = non-prime edited insertions or deletions. Source data are provided as a Source Data file.