Search-and-replace genome editing without double-strand breaks or donor DNA

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Abstract

Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2,3,4,5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay–Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.

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Fig. 1: Overview of prime editing and feasibility studies in vitro and in yeast cells.
Fig. 2: Prime editing of genomic DNA in human cells by PE1 and PE2.
Fig. 3: PE3 and PE3b systems nick the non-edited strand to increase prime editing efficiency.
Fig. 4: Targeted insertions, deletions, and all 12 types of point mutation with PE3 at seven endogenous genomic loci in HEK293T cells.
Fig. 5: Prime editing of pathogenic mutations, prime editing in primary mouse cortical neurons, and comparison of prime editing and HDR in four human cell lines.

Data availability

High-throughput sequencing data have been deposited to the NCBI Sequence Read Archive database under accession PRJNA565979. Plasmids encoding PE1, PE2 (same as PE3), and pegRNA expression vectors are available from Addgene. Previously described plasmids expressing sgRNAs are also available from Addgene, such as Addgene plasmid no. 65777.

Code availability

The script used to quantify pegRNA scaffold insertion is provided as Supplementary Note 4.

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Acknowledgements

We thank J. M. Madison for neuron cell culture advice. This work was supported by the Merkin Institute of Transformative Technologies in Healthcare, US NIH grants U01AI142756, RM1HG009490, R01EB022376, and R35GM118062, and the HHMI. A.V.A. acknowledges a Jane Coffin Childs postdoctoral fellowship. P.B.R. and A.R. acknowledge NIH T32 GM095450. A.A.S. acknowledges NIH T32 GM007726. P.J.C. and A.R. acknowledge NSF graduate fellowships. C.W. acknowledges a Damon Runyon Cancer Research Foundation fellowship (DRG-2343-18). G.A.N acknowledges a Helen Hay Whitney postdoctoral fellowship.

Author information

A.V.A. designed the research, performed experiments, analysed data, and wrote the manuscript. P.B.R., J.R.D., A.A.S., and G.A.N. performed human cell experiments and analysed data. L.W.K. and J.M.L. performed neuron experiments. P.J.C. and C.W. performed and analysed RNA-seq experiments. A.R. analysed ClinVar data. D.R.L designed and supervised the research and wrote the manuscript.

Correspondence to David R. Liu.

Ethics declarations

Competing interests

Authors through the Broad Institute have filed patent applications on prime editing. D.R.L. is a consultant and co-founder of Prime Medicine, Beam Therapeutics, Pairwise Plants, and Editas Medicine, companies that use genome editing.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Guangping Gao, Randall Platt and Fyodor Urnov for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 In vitro prime editing validation studies with fluorescently labelled DNA substrates.

a, Electrophoretic mobility shift assays with dCas9, 5′-extended pegRNAs and 5′-Cy5-labelled DNA substrates. pegRNAs 1–5 contain a 15-nt linker sequence (linker A for pegRNA 1, linker B for pegRNAs 2–5) between the spacer and the PBS, a 5-nt PBS sequence, and RT templates of 7 nt (pegRNAs 1 and 2), 8 nt (pegRNA 3), 15 nt (pegRNA 4), and 22 nt (pegRNA 5). pegRNAs are those used in e and f; full sequences are listed in Supplementary Table 2. b, In vitro nicking assays of Cas9(H840A) using 5′-extended and 3′-extended pegRNAs. Data in a, b are representative of n = 2 independent replicates. c, Cas9-mediated indel formation in HEK293T cells at HEK3 using 5′-extended and 3′-extended pegRNAs. Mean ± s.d. of n = 3 independent biological replicates. d, Overview of prime editing in vitro biochemical assays. 5′-Cy5-labelled pre-nicked and non-nicked dsDNA substrates were tested. sgRNAs, 5′-extended pegRNAs, or 3′-extended pegRNAs were pre-complexed with dCas9 or Cas9(H840A) nickase, then combined with dsDNA substrate, Superscript III M-MLV RT, and dNTPs. Reactions were allowed to proceed at 37 °C for 1 h before separation by denaturing urea PAGE and visualization by Cy5 fluorescence. e, Primer extension reactions using 5′-extended pegRNAs, pre-nicked DNA substrates, and dCas9 lead to substantial conversion to RT products. f, Primer extension reactions using 5′-extended pegRNAs as in b with non-nicked DNA substrate and Cas9(H840A) nickase. Product yields are greatly reduced by comparison to pre-nicked substrate. g, An in vitro primer extension reaction using a 3′-pegRNA generates a single apparent product by denaturing urea PAGE. The RT product band was excised, eluted from the gel, then subjected to homopolymer tailing with terminal transferase (TdT) using either dGTP or dATP. Tailed products were extended using poly-T or poly-C primers, and the resulting DNA was sequenced. Sanger traces indicate that three nucleotides derived from the pegRNA scaffold were reverse-transcribed (added as the final 3′ nucleotides to the DNA product). Note that pegRNA scaffold insertion is much rarer in mammalian cell prime editing experiments than in vitro (Extended Data Fig. 6), potentially owing to the inability of the tethered RT to access the Cas9-bound guide RNA scaffold, and/or cellular excision of mismatched 3′ ends of 3′ flaps containing pegRNA scaffold sequences. Data in eg are representative of n = 2 independent replicates. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 Cellular repair in yeast of 3′ DNA flaps from in vitro prime editing reactions.

a, Dual fluorescent protein reporter plasmids contain GFP and mCherry open reading frames separated by a target site encoding an in-frame stop codon, a +1 frameshift, or a −1 frameshift. Prime editing reactions were carried out in vitro with Cas9(H840A) nickase, pegRNA, dNTPs, and M-MLV RT, then transformed into yeast. Colonies that contain unedited plasmids produce GFP but not mCherry. Yeast colonies containing edited plasmids produce both GFP and mCherry as a fusion protein. b, Overlay of GFP and mCherry fluorescence for yeast colonies transformed with reporter plasmids containing a stop codon between GFP and mCherry (unedited negative control, top), or containing no stop codon or frameshift between GFP and mCherry (pre-edited positive control, bottom). cf, Visualization of mCherry and GFP fluorescence from yeast colonies transformed with in vitro prime editing reaction products. c, d, Stop codon correction via T•A-to-A•T transversion using a 3′-extended pegRNA (c) or a 5′-extended pegRNA (d). e, +1 frameshift correction via a 1-bp deletion using a 3′-extended pegRNA. f, −1 frameshift correction via a 1-bp insertion using a 3′-extended pegRNA. g, Sanger DNA sequencing traces from plasmids isolated from GFP-only colonies in b and GFP and mCherry double-positive colonies in c. Data in bg are representative of n = 2 independent replicates.

Extended Data Fig. 3 Prime editing of genomic DNA in human cells by PE1.

a, pegRNAs contain a spacer sequence, an sgRNA scaffold, and a 3′ extension containing an RT template (purple), which contains the edited base(s) (red), and a primer-binding site (PBS, green). The primer-binding site hybridizes to the nicked target DNA strand. The RT template is homologous to the DNA sequence downstream of the nick, with the exception of the encoded edited base(s). b, Installation of a T•A-to-A•T transversion at the HEK3 site in HEK293T cells using Cas9(H840A) nickase fused to wild-type M-MLV RT (PE1) and pegRNAs with varying PBS lengths. c, T•A-to-A•T transversion editing efficiency and indel generation by PE1 at the +1 position of HEK3 using pegRNAs containing 10-nt RT templates and PBS sequences ranging from 8 to 17 nt. d, G•C-to-T•A transversion editing efficiency and indel generation by PE1 at the +5 position of EMX1 using pegRNAs containing 13-nt RT templates and PBS sequences ranging from 9 to 17 nt. e, G•C-to-T•A transversion editing efficiency and indel generation by PE1 at the +5 position of FANCF using pegRNAs containing 17-nt RT templates and PBS sequences ranging from 8 to 17 nt. f, C•G-to-A•T transversion editing efficiency and indel generation by PE1 at the +1 position of RNF2 using pegRNAs containing 11-nt RT templates and PBS sequences ranging from 9 to 17 nt. g, G•C-to-T•A transversion editing efficiency and indel generation by PE1 at the +2 position of HEK4 using pegRNAs containing 13-nt RT templates and PBS sequences ranging from 7 to 15 nt. h, PE1-mediated +1 T deletion, +1 A insertion, and +1 CTT insertion at the HEK3 site using a 13-nt PBS and a 10-nt RT template. Sequences of pegRNAs are as in Fig. 2a (Supplementary Table 3). Editing efficiencies reflect sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 4 Evaluation of M-MLV RT variants for prime editing.

a, Abbreviations for prime editor variants used in this figure. b, Targeted insertion and deletion edits with PE1 at the HEK3 locus. ch, Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install a +2 G•C-to-C•G transversion edit at HEK3 (c), a 24-bp Flag insertion at the +1 position of HEK3 (d), a +1 C•G-to-A•T transversion edit at RNF2 (e), a +1 G•C-to-C•G transversion edit at EMX1 (f), a +2 T•A-to-A•T transversion edit at HBB (g), and a +1 G•C-to-C•G transversion edit at FANCF (h). in, Comparison of four prime editor constructs containing M-MLV variants for their ability to install the edits shown in ch in a second round of independent experiments. os, PE2 editing efficiency at five genomic loci with varying PBS lengths. o, +1 T•A-to-A•T at HEK3. p, +5 G•C-to-T•A at EMX1. q, +5 G•C-to-T•A at FANCF. r, +1 C•G-to-A•T at RNF2. s, +2 G•C-to-T•A at HEK4. Editing efficiencies reflect sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 5 Design features of pegRNA PBS and RT template sequences, and additional editing examples with PE3.

a, PE2-mediated +5 G•C-to-T•A transversion editing efficiency (blue line) at VEGFA in HEK293T cells as a function of RT template length. Indels (grey line) are plotted for comparison. The sequence below the graph shows the last nucleotide templated for synthesis by the pegRNA. G nucleotides (templated by a C in the pegRNA) are highlighted in red; RT templates that end in C should be avoided during pegRNA design to maximize prime editing efficiency. b, +5 G•C-to-T•A transversion editing and indels for DNMT1 as in a. c, +5 G•C-to-T•A transversion editing and indels for RUNX1 as in a. df, PE3-mediated transition and transversion edits at the specified positions for FANCF (d), EMX1 (e), and DNMT1 (f). Mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 6 Comparison of prime editing and base editing, and off-target editing by Cas9 and prime editors at known Cas9 off-target sites.

a, C•G-to-T•A editing efficiency at the same target nucleotides for PE2, PE3, BE2max, and BE4max at endogenous HEK3, FANCF, and EMX1 sites in HEK293T cells. b, Indel frequency from treatments in a. c, Editing efficiency of precise C•G-to-T•A edits (without bystander edits or indels) at HEK3, FANCF, and EMX1. d, Total A•T-to-G•C editing efficiency for PE2, PE3, ABEdmax, and ABEmax at HEK3 and FANCF. e, Precise A•T-to-G•C editing efficiency without bystander edits or indels at HEK3 and FANCF. f, Indel frequency from treatments in d. g, Average triplicate Cas9 nuclease editing efficiencies (indel frequencies) in HEK293T cells at four endogenous on-target sites and their 16 known top off-target sites32,33. For each on-target site, Cas9 was paired with an sgRNA or with each of four pegRNAs that recognize the same protospacer. h, Average triplicate on-target and off-target editing efficiencies and indel efficiencies (below in parentheses) in HEK293T cells for PE2 or PE3 paired with each pegRNA in g. Editing efficiencies reflect sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Off-target editing efficiencies in h reflect off-target locus modification consistent with prime editing. Mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 7 Incorporation of pegRNA scaffold sequence into target loci.

HTS data were analysed for pegRNA scaffold sequence insertion as described in Supplementary Note 4. a, Analysis for the EMX1 locus. Shown is the percentage of total sequencing reads containing one or more pegRNA scaffold sequence nucleotides within an insertion adjacent to the RT template (left); the percentage of total sequencing reads containing a pegRNA scaffold sequence insertion of the specified length (middle); and the cumulative total percentage of pegRNA insertion up to and including the length specified on the x-axis. b, As in a for FANCF. c, As in a for HEK3. d, As in a for RNF2. Mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 8 Effects of PE2, PE2-dRT, Cas9(H840A) nickase, and dCas9 on cell viability and on transcriptome-wide RNA abundance.

HEK293T cells were transiently transfected with plasmids encoding PE2, PE2(R110S/K103L), Cas9(H840A) nickase, or dCas9, together with a HEK3-targeting pegRNA plasmid. Cell viability was measured for the bulk cellular population every 24 h after transfection for 3 days using the CellTiter-Glo 2.0 assay (Promega). a, Viability, as measured by luminescence, at 1, 2, or 3 days after transfection. Mean ± s.e.m. of n = 3 independent biological replicates, each performed in technical triplicate. b, Percentage editing and indels for PE2, PE2(R110S/K103L), Cas9(H840A) nickase, or dCas9, together with a HEK3-targeting pegRNA plasmid that encodes a +5 G-to-A edit. Editing efficiencies were measured on day 3 after transfection from cells treated alongside those used for assaying viability in a. Mean ± s.d. of n = 3 independent biological replicates. ck, Analysis of cellular RNA, depleted for ribosomal RNA, isolated from HEK293T cells expressing PE2, PE2-dRT, or Cas9(H840A) nickase and a PRNP-targeting or HEXA-targeting pegRNA. RNAs corresponding to 14,410 genes and 14,368 genes were detected in PRNP and HEXA samples, respectively. ch, Volcano plot displaying the −log10 FDR-adjusted P value versus log2-fold change in transcript abundance for each RNA, comparing PE2 versus PE2-dRT with PRNP-targeting pegRNA (c), PE2 versus Cas9(H840A) with PRNP-targeting pegRNA (d), PE2-dRT versus Cas9(H840A) with PRNP-targeting pegRNA (e), PE2 versus PE2-dRT with HEXA-targeting pegRNA(f), PE2 versus Cas9(H840A) with HEXA-targeting pegRNA (g), PE2-dRT versus Cas9(H840A) with HEXA-targeting pegRNA (h). Red dots indicate genes that show twofold or more changes in relative abundance that are statistically significant (FDR-adjusted P < 0.05). ik, Venn diagrams of upregulated and downregulated transcripts (twofold change or more) comparing PRNP and HEXA samples for PE2 versus PE2-dRT (i), PE2 versus Cas9(H840A) (j), and PE2-dRT versus Cas9(H840A) (k). Values for each RNA-seq condition reflect the mean of n = 5 biological replicates. Differential expression was assessed using a two-sided t-test with empirical Bayesian variance estimation.

Extended Data Fig. 9 PE3-mediated correction of E6V-encoding HBB mutation and HEXA1278+TATC by various pegRNAs.

a, Screen of 14 pegRNAs for correction of the HBB E6V-encoding allele in HEK293T cells with PE3. All pegRNAs evaluated convert the mutant HBB allele back to wild-type HBB without the introduction of any silent PAM mutation. b, Screen of 41 pegRNAs for correction of the HEXA1278+TATC allele in HEK293T cells with PE3 or PE3b. Those pegRNAs labelled HEXAs correct the pathogenic allele by a shifted 4-bp deletion that disrupts the PAM and leaves a silent mutation. Those pegRNAs labelled HEXA correct the pathogenic allele back to wild-type. Entries ending in b use an edit-specific nicking sgRNA in combination with the pegRNA (the PE3b system). Mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 10 PE3 activity in human cell lines and comparison of PE3 and Cas9-initiated HDR.

a, Prime editing in K562 (leukaemic bone marrow), U2OS (osteosarcoma), and HeLa (cervical cancer) cells. be, Efficiency of generating the correct edit (without indels) and indel frequency for PE3 and Cas9-initiated HDR in HEK293T cells (b), K562 cells (c), U2OS cells (d), and HeLa cells (e). Each bracketed editing comparison installs identical edits with PE3 and Cas9-initiated HDR. Non-targeting controls are PE3 and a pegRNA that targets a non-target locus. (f) Control experiments with non-targeting pegRNA + PE3, and with dCas9 + sgRNA, compared with wild-type Cas9 HDR experiments confirming that ssDNA donor HDR template, a common contaminant that artificially elevates apparent HDR efficiencies, does not contribute to the HDR measurements in ad. g, Example HEK3 site allele tables from genomic DNA samples isolated from K562 cells after editing with PE3 or with Cas9-initiated HDR. Alleles were sequenced on an Illumina MiSeq and analysed using CRISPResso243. The reference HEK3 sequence from this region is at the top. Allele tables are shown for a non-targeting pegRNA negative control, a +1 CTT insertion at HEK3 using PE3, and a +1 CTT insertion at HEK3 using Cas9-initiated HDR. Allele frequencies and corresponding Illumina sequencing read counts are shown for each allele. All alleles observed with frequency ≥0.20% are shown. Mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 11 Distribution by length of pathogenic insertions, duplications, deletions, and indels in the ClinVar database.

The ClinVar variant summary was downloaded from NCBI on 15 July 2019. The lengths of reported insertions, deletions, and duplications were calculated using reference and alternate alleles, variant start and stop positions, or appropriate identifying information in the variant name. Variants that did not report any of the above information were excluded from the analysis. The lengths of reported indels (single variants that include both insertions and deletions relative to the reference genome) were calculated by determining the number of mismatches or gaps in the best pairwise alignment between the reference and alternate alleles. a, Length distribution of insertions. b, Length distribution of duplications. c, Length distribution of deletions. d, Length distribution of indels.

Supplementary information

Supplementary Information

The Supplementary Information file contains: a Supplementary Discussion that provides additional background and descriptions of the data; Supplementary Tables 1-5 listing activity data and short DNA and RNA sequences; Supplementary Sequences 1-3 that contain DNA and protein sequences of key constructs; Supplementary Notes 1-4 that contain FACS information, cloning methods, and scripts; Supplementary Figure 1 that contains original gel image data, and Supplementary References

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Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature (2019) doi:10.1038/s41586-019-1711-4

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