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Increasing the efficiency and precision of prime editing with guide RNA pairs

Abstract

The recently reported prime editor (PE) can produce all types of base substitution, insertion and deletion, greatly expanding the scope of genome editing. However, improving the editing efficiency and precision of PE represents a major challenge. Here, we report an approach termed the homologous 3′ extension mediated prime editor (HOPE). HOPE uses paired prime editing guide RNAs (pegRNAs) encoding the same edits in both sense and antisense DNA strands to achieve high editing efficiency in human embryonic kidney 293T cells as well as mismatch repair-deficient human colorectal carcinoma 116 cells. In addition, we found that HOPE shows greatly improved product purity compared to the original PE3 system. We envision that this enhanced tool could broaden both fundamental research and therapeutic applications of prime editing.

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Fig. 1: HOPE facilitates efficient base substitutions via paired pegRNAs in human cells.
Fig. 2: Characterizing the influence of PAM-in distances on HOPE editing capability.
Fig. 3: HOPE demonstrates improved editing precision compared to PE3.
Fig. 4: HOPE mediates effective and precise insertions and deletions.
Fig. 5: HOPE mediates effective and precise editing in HCT116 cells.
Fig. 6: Off-target effect examination of HOPE.

Data availability

All data generated for this study have been deposited in the NCBI Gene Expression Omnibus under accession number GSE171470. Source data are provided with this paper.

Code availability

The custom code and scripts have been deposited into GitHub (https://github.com/James-Hao/HOPE).

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Acknowledgements

We thank D. Xu for kindly offering us the HCT116 cell line and its culture methods, and Z. Lei for advice on targeted amplicon sequencing. We thank the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with fragment analysis (quality control of DNA samples) using Agilent 4150 TapeStation system. We also thank the flow cytometry core at National Center for Protein Sciences at Peking University, particularly Y. Guo, for technical help. We carried out data analysis on the High-Performance Computing Platform at the School of Life Sciences, Peking University. This work was supported by the National Natural Science Foundation of China (grant nos. 21825701, 91953201 and 31861143026 to C.Y.) and Ministry of Science and Technology of China (grant nos. 2019YFA0110900 and 2019YFA0802201 to C.Y.).

Author information

Authors and Affiliations

Authors

Contributions

C.Y., Y.Z. and J.L. conceived and guided the research. Y.Z. and J.L. designed and performed the experiments. H.W. and H.M analyzed the sequencing data. Q.Z and Y.Y assisted with the experiments on plasmid preparation, genome DNA extraction and target sequences amplification. P.R.C. participated in the design and interpretation of key experiments. Y.Z., J.L., H.W. and C.Y. wrote the manuscript. All the authors commented on and approved the paper.

Corresponding author

Correspondence to Chengqi Yi.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Chemical Biology thanks Jia Chen, Keiichiro Suzuki and other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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

Extended data

Extended Data Fig. 1 Schematic representation of vectors and sequences.

a, Schematic representation of the original PE vectors. b, Schematic representation of HOPE vectors. c, d, Representative sequences of human PDCD1 (c) and METTL3 (d) loci. Blue and orange arrows indicate the S-pegRNA and AS-pegRNA sequences, respectively.

Extended Data Fig. 2 Undesired indel features of HOPE and the original PE systems.

a, b, Undesired insertion and deletion ratios of HOPE and the optimal PE3 at the EMX1 (a) and FANCF (b) loci. The PAM-in distances of HOPE are 7 nt and 14 nt at the FANCF and EMX1 loci, respectively. c, d, Indel size analysis of PE2, PE3 and HOPE at the EMX1 (c) and FANCF (d) loci. The orange and green lines indicate the undesired insertions and deletions, respectively. e, f, Representative allele tables from samples treated with PE2, HOPE and optimal PE3 at the FANCF locus (e) and EMX1 locus (f). All alleles observed with frequencies ≥ 0.02% are shown. g, h, Heatmaps reflect PE2 undesired insertion ratio and distribution of the EMX1 (g) and FANCF (h) loci. The nick sites induced by S-pegRNAs are set as position 0. i, j, Undesired deletion ratio and length range induced by PE2 at the FANCF (i) and EMX1 (j) sites. Each black line represents a single deletion event. All types of deletions are shown for each sample.

Source data

Extended Data Fig. 3 Optimizing PBS for HOPE.

a-e, Comparison of the desired editing efficiencies under the diverse PBS Tm/length conditions at five genomic loci. The desired editing types and positions were referred to the sense DNA strand. The desired editing efficiencies of S-PE2, AS-PE2 and HOPE are calculated by CRISPResso2. n = 3 independently biological replicates in (a-d), while n = 2 independently biological replicates in (e), mean with s.d. f, Standardized efficiencies (Z-Score normalization) of HOPE under diverse PBS Tm conditions at the five tested sites. Locally estimated scatterplot smoothing (LOESS) regression models are used to fit a smooth curve. Data shown as mean ± s.d. n = 3, 9, 19, 6, 17, 5, 6 and 3 in the ‘55-50’, ‘49-45’, ‘44-40’, ‘39-35’, ‘34-30’, ‘29-25’, ‘24-20’ and ‘19-15’ groups, respectively.

Source data

Extended Data Fig. 4 Optimizing RT templates for HOPE.

a, Diagram of total RT length (TL), downstream of edits length (DL) of 3’ extensions for pegRNAs. b, Diagram of homologous length of 3’ extensions (brown numbers in heatmaps) for pegRNAs. c-i, Editing frequencies of S-PE2, AS-PE2s and their combinations as HOPE with diverse RT template lengths, at PDCD1 site2 locus for +8 C to G substitution (c); ABE site21 locus for +8 C to A substitution (d); EMX1 locus for +14 C to G substitution (e); PCIF1 locus for +9-10 AA to GC substitutions (f); FANCF locus for +11 C to G substitution (g); METTL3 locus for +10 A to C substitution (h), HEK site4 locus for +7 TGA insertions (i). The desired editing types and positions were referred to the sense DNA strand.

Source data

Extended Data Fig. 5 Examining Off-target effect of HOPE.

a, b, Off-target sites predicted by Cas-OFFinder with mismatch numbers ≤ 3 for S-pegRNA (a) and AS-pegRNA (b) of the HEK site3 locus. Bar graph showing the insertion or indel ratio at the predicted off-target editing sites, or a ± 25 bp region surrounding the off-target sites. BG: background substitution/indel level. Data shown as mean ± s.d. n = 3 independently biological replicates. P-values were calculated by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Tables 1–6 and Note 1.

Reporting Summary

Supplementary Table 1

Primer sequences for off-target examination.

Source data

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Zhuang, Y., Liu, J., Wu, H. et al. Increasing the efficiency and precision of prime editing with guide RNA pairs. Nat Chem Biol 18, 29–37 (2022). https://doi.org/10.1038/s41589-021-00889-1

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