Abstract
Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.
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Data availability
The data that support the findings of current study are available within this manuscript and under National Center for Biotechnology Information BioProject accession no. PRJNA743217. All deep-sequencing data in this study are provided in Supplementary Tables 1, 3 and 8. Source data are provided with this paper.
Code availability
Bioinformatics codes have been deposited in the GitHub repository (https://github.com/skqxys/ GRAND-editing).
References
Orthwein, A. et al. A mechanism for the suppression of homologous recombination in G1 cells. Nature 528, 422–426 (2015).
Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).
Yin, H., Kauffman, K. J. & Anderson, D. G. Delivery technologies for genome editing. Nat. Rev. Drug Discov. 16, 387–399 (2017).
Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
Chapman, J. R., Taylor, M. R. G. & Boulton, S. J. Playing the end game: DNA double-strand break repair pathway choice. Mol. Cell 47, 497–510 (2012).
Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).
Maresca, M., Lin, V. G., Guo, N. & Yang, Y. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res. 23, 539–546 (2013).
Suzuki, K. et al. Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction. Cell Res. 29, 804–819 (2019).
Li, Dali et al. Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration. Preprint at BioRxiv https://doi.org/10.1101/2021.03.18.435908 (2021).
Nami, F. et al. Strategies for in vivo genome editing in nondividing cells. Trends Biotechnol. 36, 770–786 (2018).
Jiang, T. T., Zhang, X. O., Weng, Z. P. & Xue, W. Deletion and replacement of long genomic sequences using prime editing. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-01026-y (2021).
Landrum, M. J. et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 42, D980–D985 (2014).
Shastry, B. S. SNPs in disease gene mapping, medicinal drug development and evolution. J. Hum. Genet. 52, 871–880 (2007).
McClellan, J. & King, M. C. Genetic heterogeneity in human disease. Cell 141, 210–217 (2010).
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365, 382–386 (2019).
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science https://doi.org/10.1126/science.aaf8729 (2016).
Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021).
Zhao, D. D. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat. Biotechnol. 39, 35–40 (2021).
Chen, L. et al. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat. Commun. 12, 1384 (2021).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Liu, Y., Kao, H. I. & Bambara, R. A. Flap endonuclease 1: a central component of DNA metabolism. Annu. Rev. Biochem. 73, 589–615 (2004).
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 (2021).
Dai, X. et al. One-step generation of modular CAR-T cells with AAV-Cpf1. Nat. Methods 16, 247–254 (2019).
Fry, D. W. et al. Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol. Cancer Ther. 3, 1427–1438 (2004).
DiRocco, D. P. et al. CDK4/6 inhibition induces epithelial cell cycle arrest and ameliorates acute kidney injury. Am. J. Physiol. Ren. Physiol. 306, F379–F388 (2014).
Scott, S. J., Suvarna, K. S. & D’Avino, P. P. Synchronization of human retinal pigment epithelial-1 cells in mitosis. J. Cell Sci. 133, jcs247940 (2020).
Harper, J. V. Synchronization of cell populations in G1/S and G2/M phases of the cell cycle. Methods Mol. Biol. 296, 157–166 (2005).
Jin, S. et al. Genome-wide specificity of prime editors in plants. Nat. Biotechnol. 39, 1292–1299 (2021).
Gao, R. et al. Genomic and transcriptomic analyses of prime editing guide RNA-independent off-target effects by prime editors. CRISPR J. https://doi.org/10.1089/crispr.2021.0080 (2022).
Menendez-Arias, L. Mutation rates and intrinsic fidelity of retroviral reverse transcriptases. Viruses 1, 1137–1165 (2009).
Lin, Q. P. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39, 923–927 (2021).
Zhuang, Y. et al. Increasing the efficiency and precision of prime editing with guide RNA pairs. Nat. Chem. Biol. https://doi.org/10.1038/s41589-021-00889-1 (2021).
Choi, J. et al. Precise genomic deletions using paired prime editing. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-01025-z (2021).
Kutner, R. H., Zhang, X. Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495–505 (2009).
Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J. & Elledge, S. J. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. PNAS 102, 13212–13217 (2005).
Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).
Chen, S. F., Zhou, Y. Q., Chen, Y. R. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, 884–890 (2018).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, Heng et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-01039-7 (2021).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Acknowledgements
This work is kindly supported by National Key R&D Program of China (2019YFA0802801 and 2018YFA0801401, to H.Y.), the National Natural Science Foundation of China (31871345 and 32071442 to H.Y., 31972936 to Y.Z.), Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University (TFJC2018004), the Fundamental Research Funds for the Central Universities (to H.Y. and Y.Z.), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2020-PT320-004), Applied Basic Frontier Program of Wuhan City (2020020601012216 to H.Y.), Hubei Health Commission Young Investigator award (to H.Y.) and startup funding from Wuhan University (to H.Y and Y.Z). We thank staff at the core facility of the Medical Research Institute at Wuhan University for their technical support. We thank Y. Zhou, S. Li (Wuhan University) and Q. Zhang (Huazhong University of Science and Technology) for their technical assistance.
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Contributions
H.Y. conceived, designed and managed the project. J.W., Z.H., G.W. and R.Z. performed most experiments with the help of X.L., C.Z. and H.Q. Y.Z. provided conceptual advice and edited the manuscript. P.G. and J.D. performed bioinformatic analysis. J.W. and H.Y. analyzed the data. H.Y. wrote the paper with input from all authors.
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H.Y., Y.Z., J.W., Z.H., G.W. and R.Z. have filed patent applications on GRAND editing through Wuhan University. The other authors declare no competing interests.
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Nature Methods thanks Tao Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Supplementary data for targeted insertion of large DNA fragment at EGFP site.
a, The imperfect events generated from 101 bp insertion by GRAND editing were mainly classified as five types. b, Illustration of deep sequencing data for targeted insertion of 458, 600, 767 and 1085 bp DNA fragments at EGFP site. The edited regions were amplified by PCR, and deep sequencing were performed. The diagram shows the analysis method of deep sequencing data.
Extended Data Fig. 2 Targeted insertion of functional fragments at EGFP site.
a, The 458 bp P2A-bsd gene was in-frame inserted into EGFP site. The HEK293T-EGFP cells were cultured with blasticidin at 24 h after transfection of GRAND editing. Eight days later, the editing frequency was evaluated by TA cloning and subsequently Sanger sequencing of 23 individual clones. b,c, The 315 bp EGFP coding sequence was in-frame inserted into disturbed EGFP site (341-647) to restore the function of EGFP gene (n = 3 independent experiments). b, Representative images of precisely edited cells (5 days after transfection). The edited cells that restored EGFP fluorescence were pointed by blue arrows. Bars, 500 μm. c, The edited cells with active EGFP were sorted by flow cytometry, and EGFP site was amplified and the PCR product was visualized in 1.5% agarose gel. The EGFP ctrl (line 4) was PCR product amplified from full-length EGFP plasmid.
Extended Data Fig. 3 GRAND editing achieved efficient and accurate insertion of short fragment.
a, TIDE analysis can measure efficiency of DNA modification within 50 bp range. Insertion of 20 bp or 66 bp with a 53 bp deletion cause -33 bp or +13 bp change, respectively. The editing efficiencies were determined by TIDE analysis. b, Semi-quantitative analysis of 87 bp insertion (with a 53 bp deletion) by agarose gel. c, The efficiencies of accurate insertion of short fragment and imperfect editing were measured by deep sequencing. Mean ± s.d. of n = 3 independent biological replicates.
Extended Data Fig. 4 Supplementary data for targeted insertion of large DNA fragment at other endogenous loci.
Semi-quantitative analysis of 150 bp insertion by TAE agarose gel. The bands of predicted size were marked with red arrows while the unexpected bands were marked with green arrows.
Extended Data Fig. 5
Illustration of the strategies of real-time qPCR and In-Out PCR to quantify insertion efficiency. a, The edited samples mainly include two types of sequences. Two pairs of qPCR primers were designed to amplify the WT strands or edited strands at the LSP1 locus, respectively. The sequence between paired pegRNAs was showed in purple, and the insertion fragment was show in red. b, The WT and edited sequences were designed and synthesized. These two fragments were serially diluted from 10-2 to 10-8 ng/μL to serve as templates for standard curve. c, Two standard curves were generated by real-time qPCR using primers and templates described above. The standard curve reflects the correlation between DNA copy numbers and quantification cycle (Cq). d, Diagram of In-Out PCR for detecting inserted fragment. Two pairs of primers (P1/P2: green arrows; P3/P4 red arrows) were designed to amplify the WT and edited genomes. For the WT genome, the P1,P2 and P4 but not P3 could bind to the genome sequence. For the edited genome, the two pairs of primers can amplify both WT and edited sequences. e, Detection of the precise insertion of 150 bp fragments at five endogenous loci by In-Out PCR. The expected products of P1/P2 and P3/P4 were pointed by green arrows and red arrows, respectively (n = 2 independent experiments).
Extended Data Fig. 6 Sequencing data for targeted insertion of large DNA fragment at other endogenous loci.
a, The pie chart reflects that the 150 bp insertion at HEK4 site mainly has 3 types of imperfect editing events. b, Detection of precise insertion and imperfect editing events in endogenous loci by TA cloning/Sanger sequencing of 16 to 24 individual clones. c, Sequence analysis of the 250 bp insertion at VEGFA and PSEN1 sites by TA cloning and subsequently Sanger sequencing of 20 to 24 individual clones. Mean ± s.d. of n = 3 independent biological replicates.
Extended Data Fig. 7 Comparison of the efficiencies of accurate 150 bp insertion using GRAND editing and PE3 at five endogenous loci.
a, Detection of the accurate 150 bp insertion at five sites edited by GRAND or PE3. The target regions were amplified and the PCR products were digested by HindIII restriction enzyme. The digested products were displayed by 2% TAE agarose. The digested products were indicated by the red arrows. The predicted sizes of digestion products with precise editing are listed below the image of agarose gel. b, Detecting the accurate 150 bp insertion and imperfect events of GRAND or PE3 by deep sequencing. Mean ± s.d. of n = 3 independent biological replicates.
Extended Data Fig. 8 Comparison of the insertion frequencies of large DNA fragments with different lengths of complementary base pairs.
a, Insertion of 458 or 600 bp DNA fragments at EGFP site with 60 or 200 bp complementary base pairs. b, Insertion of 767 or 1085 bp DNA fragments at EGFP site with 50 or 200 bp complementary base pairs. dGRAND: dead Cas9-RT + dual pegRNAs, used as control for deep sequencing analysis; GRAND: nick Cas9-RT + dual pegRNAs. Mean ± s.d. of n = 3 independent biological replicates.
Extended Data Fig. 9 Paired pegRNAs with fully active Cas9 nuclease-reverse transcriptase (aPE) mainly induced deletion between two double stranded breaks.
a, The diagram indicates the editing outcome of fully active Cas9 nuclease version of GRAND editing (aPE). b, Insertion of 87 or 101 bp using GRAND editing or aPE. The editing outcomes were measured by TAE agarose gel (n = 3 independent experiments). c, The Sanger sequencing result of aPE completely aligned with WT sequence with a 53 bp deletion between two double stranded breaks. d, Insertion of 150 bp foreign DNA fragments accompanied with deletion of genomic DNA by GRAND editing or aPE. The target sites were amplified using primers that bound to adjacent genomic regions. The expected precise editing bands were pointed by red arrows. e, All of the edited bands were purified by gel electrophoresis, and deep sequencing analysis was performed. Mean ± s.d. of n = 3 independent biological replicates expect VEGFA-del 348 bp in aPE.
Extended Data Fig. 10 Measurement of the activity of GRAND editing at predicted off-target sites and usage of different transfection reagents to deliver GRAND editing in HEK293T cells.
a, The predicted off-target sites of single pegRNA were selected using Cas-OFFinder, and amplicon sequencing was performed. b, To determine efficiencies of 150 bp insertion using four transfection reagents by real-time qPCR. Mean ± s.d. of n = 3 independent biological replicates.
Supplementary information
Supplementary Information
Supplementary Tables 2–4, 6 and 7, Supplementary Sequences 1–3, Supplementary Notes 1 and 2, Supplementary Protocol and Supplementary Figs. 1 and 2
Supplementary Table 1
Deep-sequencing data of EGFP site in this study.
Supplementary Table 5
Deep-sequencing data of off-target sites in this study.
Supplementary Table 8
Deep-sequencing data of endogenous loci in this study.
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Wang, J., He, Z., Wang, G. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat Methods 19, 331–340 (2022). https://doi.org/10.1038/s41592-022-01399-1
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DOI: https://doi.org/10.1038/s41592-022-01399-1
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