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

Abstract

Most genetic variants that contribute to disease¹ are challenging to correct efficiently and without excess byproducts^2-5. 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.

Extended Data Figure 1.. In vitro prime editing validation studies with fluorescently labeled DNA substrates.

(a) Electrophoretic mobility shift assays with dCas9, 5’-extended pegRNAs and 5’-Cy5-labeled DNA substrates. pegRNAs 1 through 5 contain a 15-nt linker sequence (linker A for pegRNA 1, linker B for pegRNAs 2 through 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. Values and error bars reflect mean±s.d. of n=3 independent biological replicates. (d) Overview of prime editing in vitro biochemical assays. 5’-Cy5-labeled 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 hour prior to 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 significant 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 by 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 in mammalian cell prime editing experiments, pegRNA scaffold insertion is much rarer than in vitro (Extended Data Fig. 6), potentially due to the inability of the tethered reverse transcriptase 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 (e-g) are representative of n=2 independent replicates. For gel source data, see Supplementary Figure 1.

Extended Data Figure 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 reverse transcriptase, 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). (c-f) Visualization of mCherry and GFP fluorescence from yeast colonies transformed with in vitro prime editing reaction products. (c) Stop codon correction via T•A-to-A•T transversion using a 3’-extended pegRNA or (d) a 5’-extended pegRNA. (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 (b-g) are representative of n=2 independent replicates.

Extended Data Figure 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 a reverse transcription (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 reverse transcriptase (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 a PBS sequences ranging from 8-17 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 a PBS sequences ranging from 9-17 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 a pBs sequences ranging from 8-17 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 a PBS sequences ranging from 9-17 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 a PBS sequences ranging from 7-15 nt. (h) PE1-mediated +1 T deletion, +1 A insertion, and +1 CTT insertion at the HEK3 site using a 13-nt PBS and 10-nt RT template. Sequences of pegRNAs are those used in Fig. 2a (see 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. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

Extended Data Figure 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. (c-h) Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install (c) a +2 G•C-to-C•G transversion edit at HEK3, (d) a 24-bp FLAG insertion at the +1 position of HEK3, (e) a +1 C•G-to-A•T transversion edit at RNF2, (f) a +1 G•C-to-C•G transversion edit at EMX1, (g) a +2 T•A-to-A•T transversion edit at HBB, and (h) a +1 G•C-to-C•G transversion edit at FANCF. (i-n) Comparison of four prime editor constructs containing M-MLV variants for their ability to install the edits shown in (c-h) in a second round of independent experiments. (o-s) 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. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

Extended Data Figure 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 (gray 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 efficiencies. (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). PE3-mediated transition and transversion edits at the specified positions for (d) FANCF, (e) EMX1, and (f) DNMT1. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

Extended Data Figure 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 sites^,. 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. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

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

HTS data were analyzed for pegRNA scaffold sequence insertion as described in Supplementary Note 4. (a) Analysis for the EMX1 locus. Shown is the % 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. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

Extended Data Figure 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 hours post-transfection for 3 days using the CellTiter-Glo 2.0 assay (Promega). (a) Viability, as measured by luminescence, at 1, 2, or 3 days post-transfection. Values and error bars reflect mean±s.e.m. of n=3 independent biological replicates, each performed in technical triplicate. (b) Percent 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 post-transfection from cells treated alongside of those used for assaying viability in (a). Values and error bars reflect mean±s.d. of n=3 independent biological replicates. (c-k) 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. (c-h) Volcano plot displaying the −log₁₀ FDR-adjusted p-value vs. log₂-fold change in transcript abundance for each RNA, comparing (c) PE2 vs. pE2-dRT with PRNP-targeting pegRNA, (d) PE2 vs. Cas9 H840A with PRNP-targeting pegRNA, (e) PE2-dRT vs. Cas9 H840A with PRNP-targeting pegRNA, (f) PE2 vs. PE2-dRT with HeXa-targeting pegRNA, (g) PE2 vs. Cas9 H840A with HEXA-targeting pegRNA, (h) PE2-dRT vs. Cas9 H840A with HEXA-targeting pegRNA. Red dots indicate genes that show ≥2-fold change in relative abundance that are statistically significant (FDR-adjusted p < 0.05). (i-k) Venn diagrams of upregulated and downregulated transcripts (≥2-fold change) comparing PRNP and HEXA samples for (i) PE2 vs PE2-dRT, (j) PE2 vs. Cas9 H840A, and (k) PE2-dRT vs. Cas9 H840A. Values for each RNA-seq condition reflect the mean n=5 biological replicates. Differential expression was assessed using a two-sided t-test with empirical Bayesian variance estimation.

Extended Data Figure 9.. PE3-mediated HBB E6V correction and HEXA 1278+TATC correction by various pegRNAs.

(a) Screen of 14 pegRNAs for correction of the HBB E6V allele in HEK293T cells with PE3. All pegRNAs evaluated convert the HBB E6V allele back to wild-type HBB without the introduction of any silent PAM mutation. (b) Screen of 41 pegRNAs for correction of the HEXA 1278+TATC allele in HEK293T cells with pE3 or PE3b. Those pegRNAs labeled HEXAs correct the pathogenic allele by a shifted 4-bp deletion that disrupts the PAM and leaves a silent mutation. Those pegRNAs labeled 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). Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

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

(a) Prime editing in K562 (leukemic bone marrow), U2OS (osteosarcoma), and HeLa (cervical cancer) cells. Efficiency of generating the correct edit (without indels) and indel frequency for PE3 and Cas9-initiated HDR in (b) HEK293T cells, (c) K562 cells, (d) U2OS cells, and (e) HeLa cells. 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 (a-d). (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 analyzed with CRISPResso2. 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. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

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

The ClinVar variant summary was downloaded from NCBI July 15, 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.

Figure 1.. Overview of prime editing and feasibility studies in vitro and in yeast cells.

(a) The 75,122 known pathogenic human genetic variants in ClinVar (accessed July, 2019), classified by type. (b) A prime editing complex consists of a prime editor (PE) protein containing an RNA-guided DNA-nicking domain, such as Cas9 nickase, fused to a reverse transcriptase domain and complexed with a prime editing guide RNA (pegRNA). The PE:pegRNA complex enables a variety of precise DNA edits at a wide range of positions. (c) The PE:pegRNA complex binds the target DNA and nicks the PAM-containing strand. The resulting 3’ end hybridizes to the primer-binding site, then primes reverse transcription of new DNA containing the desired edit using the RT template of the pegRNA. Equilibration between the edited 3’ flap and the unedited 5’ flap, cellular 5’ flap cleavage and ligation, and DNA repair results in stably edited DNA. (d) In vitro primer extension assays with 5’-extended pegRNAs, pre-nicked dsDNA substrates containing 5’-Cy5 labeled PAM strands, dCas9, and a commercial M-MLV RT variant (RT, Superscript III). dCas9 was complexed with pegRNAs, then added to DNA substrates along with the indicated components. After 1 hour, reactions were analyzed by denaturing PAGE, visualizing Cy5 fluorescence. (e) Primer extension assays performed as in (d) using 3’-extended pegRNAs pre-complexed with dCas9 or Cas9 H840A nickase, and pre-nicked or non-nicked dsDNA substrates. (f) Yeast colonies transformed with GFP–mCherry fusion reporter plasmids edited in vitro with pegRNAs, Cas9 nickase, and RT. Plasmids containing nonsense or frameshift mutations between GFP and mCherry were edited with pegRNAs that restore mCherry translation via transversion, 1-bp insertion, or 1-bp deletion. GFP and mCherry double-positive cells (yellow) reflect successful editing. Images in (d-f) are representative of n=2 independent replicates. For gel source data, see Supplementary Figure 1.

Figure 2.. Prime editing of genomic DNA in human cells by PE1 and PE2.

(a) Use of an engineered M-MLV reverse transcriptase (D200N, L603W, T306K, W313F, T330p) in PE2 substantially improves prime editing efficiencies at five genomic sites in HEK293T cells, and small insertion and small deletion edits at HEK3. (b) PE2 editing efficiencies with varying RT template lengths at five genomic sites in HEK293T cells. Editing efficiencies reflect sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

Figure 3.. PE3 and PE3b systems nick the non-edited strand to increase prime editing efficiency.

(a) Overview of prime editing by PE3. After initial synthesis of the edited strand, 5’ flap excision leaves behind a DNA heteroduplex containing one edited strand and one non-edited strand. Mismatch repair resolves the heteroduplex to give either edited or non-edited products. Nicking the non-edited strand favors repair of that strand, resulting in preferential generation of duplex DNA containing the desired edit. (b) The effect of complementary strand nicking on prime editing efficiency and indel formation. “None” refers to PE2 controls, which do not nick the complementary strand. (c) Comparison of editing efficiencies with PE2, PE3, and PE3b (edit-specific complementary strand nick). Editing efficiencies reflect sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

Figure 4.. Targeted insertions, deletions, and all 12 types of point mutations with PE3 at seven endogenous genomic loci in HEK293T cells.

(a) All 12 types of single-nucleotide edits from position +1 to +8 of the HEK3 site using a 10-nt RT template, counting the first nucleotide following the pegRNA-induced nick as position +1. (b) Long-range PE3 edits at HEK3 using a 34-nt RT template. (c-e) PE3-mediated transition and transversion edits at the specified positions for (c) RNF2, (d) RUNX1, and (e) VEGFA. (f) Targeted 1- and 3-bp insertions, and 1- and 3-bp deletions with PE3 at seven endogenous genomic loci. (g) Targeted precise deletions of 5-80 bp at HEK3. (h) Combination edits at three endogenous genomic loci. Editing efficiencies reflect sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Values and error bars reflect mean±s.d. of n=3 independent biological replicates.

Figure 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.

(a) Installation (via T•A-to-A•T transversion) and correction (via A•T-to-T•A transversion) of the pathogenic E6V mutation in HBB in HEK293T cells. Correction either to wild-type HBB, or to HBB containing a PAM-disrupting silent mutation, is shown. (b) Installation (via 4-bp insertion) and correction (via 4-bp deletion) of the pathogenic HEXA 1278+TATC allele in HEK293T cells. Correction either to wild-type HEXA, or to HEXA containing a PAM-disrupting silent mutation, is shown. (c) Installation of the protective G127V variant in PRNP in HEK293T cells via G•C-to-T•A transversion. (d) Installation of a G•C-to-T•A transversion in DNMT1 of mouse primary cortical neurons using a split-intein PE3 lentivirus system (see Methods). Sorted values reflect editing or indels from GFP-positive nuclei, while unsorted values are from all nuclei. (e) PE3 editing and indels or (f) Cas9-initiated HDR editing and indels at endogenous genomic loci in HEK293T, K562, U2OS, and HeLa cells. (g) Targeted insertion of a His₆ tag (18 bp), FLAG epitope tag (24 bp), or extended LoxP site (44 bp) in HEK293T cells by PE3. Editing efficiencies reflect sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting, except where specified in (e). Values and error bars reflect mean±s.d. of n=3 independent biological replicates.