CoChat
Sign Up
Cited by 273CRISPR and Genetic EngineeringRNA regulation and diseaseVirus-based gene therapy research

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

Open access
Liu, Pengpeng, Liang, Shun-Qing, Zheng, Chunwei, Mintzer, Esther, Zhao, Yan G., Ponnienselvan, Karthikeyan, Mir, Aamir, Sontheimer, Erik J. +4 more·2021·Nature Communications
DOI 10.1038/s41467-021-22295-wPMC PMC8035190

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41467-021-22295-w

Cites

9 of 9 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • For insertions or deletions of 10 base pairs or more, the RT should be extended by at least 25 nucleotides.

    “If the goal is to insert or delete larger portions of DNA (from 10 base pairs onwards) include an extended RT of at least 25 nucleotides47”

    From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [47]

    The Results (Fig. 1 and Fig. 2; page unavailable) report targeted deletions of 47 and 32 bp and an 18-bp insertion, supporting the use of prime editing for changes of at least 10 bp. The paper does not state that these edits require, or should use, an RT template of at least 25 nucleotides. (LLM, conf=0.78)

    Experimental — accuracy not yet measured

  • Extending the RT part of the pegRNA 10 nucleotides beyond the edit of interest is optimal for most point mutations and indels.

    “Extend the RT-part of the pegRNA 10 nucleotides beyond your edit of interest. As this has been found to be optimal for most point mutations and indels.”

    From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [47]

    The Introduction explains that a pegRNA encodes an RT template, and the Results compare prime-editor performance for point mutations, deletions, and insertions. Neither passage evaluates RT-template extension length or says that extending it 10 nucleotides beyond the edit is optimal. Searches for that length and for RT-template optimization found no supporting passage in our copy. (LLM, conf=0.74)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 41

  1. [2]Panier S Boulton SJ Double-strand break repair: 53BP1 comes into focus Nat. Rev. Mol. Cell Biol. 2014 15 7 18 10.1038/nrm3719 24326623 · DOI
  2. [3]Suzuki K In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration Nature 2016 540 144 149 10.1038/nature20565 27851729 PMC5331785 · DOI · Paper page
  3. [4]Pickar-Oliver A Gersbach CA The next generation of CRISPR-Cas technologies and applications Nat. Rev. Mol. Cell Biol. 2019 20 490 507 10.1038/s41580-019-0131-5 31147612 PMC7079207 · DOI · Paper page
  4. [5]Rees HA Liu DR Base editing: precision chemistry on the genome and transcriptome of living cells Nat. Rev. Genet. 2018 19 770 788 10.1038/s41576-018-0059-1 30323312 PMC6535181 · DOI · Paper page
  5. [6]Anzalone AV Search-and-replace genome editing without double-strand breaks or donor DNA Nature 2019 576 149 157 10.1038/s41586-019-1711-4 31634902 PMC6907074 · DOI · Paper page
  6. [8]Schene IF Prime editing for functional repair in patient-derived disease models Nat. Commun. 2020 11 5352 10.1038/s41467-020-19136-7 33097693 PMC7584657 · DOI
  7. [9]Geurts, M. H. et al. Evaluating CRISPR-based Prime Editing for cancer modeling and CFTR repair in intestinal organoids. bioRxiv 2020.2010.2005.325837 (2020). PMC8356249 34373320
  8. [10]Liu Y Efficient generation of mouse models with the prime editing system Cell Discov. 2020 6 27 10.1038/s41421-020-0165-z 32351707 PMC7186222 · DOI
  9. [11]Aida, T. et al. Prime editing primarily induces undesired outcomes in mice. bioRxiv 2020.2008.2006.239723 (2020).
  10. [12]Wu Y Highly efficient therapeutic gene editing of human hematopoietic stem cells Nat. Med. 2019 25 776 783 10.1038/s41591-019-0401-y 30911135 PMC6512986 · DOI · Paper page
  11. [13]Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol.36, 843–846 (2018). PMC6126947 29813047
  12. [14]Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol.36, 888–893 (2018). PMC6130889 29969439
  13. [15]Song C-Q Adenine base editing in an adult mouse model of tyrosinaemia Nat. Biomed. Eng. 2020 4 125 130 10.1038/s41551-019-0357-8 31740768 PMC6986236 · DOI · Paper page
  14. [17]Ray M Tang R Jiang Z Rotello VM Quantitative tracking of protein trafficking to the nucleus using cytosolic protein delivery by nanoparticle-stabilized nanocapsules Bioconjugate Chem. 2015 26 1004 1007 10.1021/acs.bioconjchem.5b00141 PMC4743495 26011555 · DOI
  15. [18]Makkerh JP Dingwall C Laskey RA Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids Curr. Biol. 1996 6 1025 1027 10.1016/S0960-9822(02)00648-6 8805337 · DOI
  16. [20]Kleinstiver BP Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition Nat. Biotechnol. 2015 33 1293 1298 10.1038/nbt.3404 26524662 PMC4689141 · DOI · Paper page
  17. [21]Certo MT Tracking genome engineering outcome at individual DNA breakpoints Nat. Methods 2011 8 671 676 10.1038/nmeth.1648 21743461 PMC3415300 · DOI
  18. [22]Mir A Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing Nat. Commun. 2018 9 2641 10.1038/s41467-018-05073-z 29980686 PMC6035171 · DOI
  19. [23]Iyer, S. et al. Efficient homology-directed repair with circular ssDNA donors. bioRxiv 864199 (2019). PMC9595650 36070530
  20. [24]Dean M Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study Science 1996 273 1856 1862 10.1126/science.273.5283.1856 8791590 · DOI
  21. [25]Liu R Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection Cell 1996 86 367 377 10.1016/S0092-8674(00)80110-5 8756719 · DOI · Paper page
  22. [26]Samson M Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene Nature 1996 382 722 725 10.1038/382722a0 8751444 · DOI
  23. [27]Bolukbasi MF Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing Nat. Commun. 2018 9 4856 10.1038/s41467-018-07310-x 30451839 PMC6242970 · DOI
  24. [28]Loring HS Flotte TR Current status of gene therapy for alpha-1 antitrypsin deficiency Expert Opin. Biol. Ther. 2015 15 329 336 10.1517/14712598.2015.978854 25363251 · DOI
  25. [29]Carlson JA Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice J. Clin. Invest. 1989 83 1183 1190 10.1172/JCI113999 2784798 PMC303805 · DOI
  26. [31]Liu F Song Y Liu D Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA Gene Ther. 1999 6 1258 1266 10.1038/sj.gt.3300947 10455434 · DOI
  27. [32]Zucman-Rossi J Villanueva A Nault JC Llovet JM Genetic landscape and biomarkers of hepatocellular carcinoma Gastroenterology 2015 149 1226 1239 10.1053/j.gastro.2015.05.061 26099527 · DOI · Paper page
  28. [33]Liu P A functional mammalian target of rapamycin complex 1 signaling is indispensable for c-Myc-driven hepatocarcinogenesis Hepatology 2017 66 167 181 10.1002/hep.29183 28370287 PMC5481473 · DOI
  29. [34]Marquardt JU Andersen JB Thorgeirsson SS Functional and genetic deconstruction of the cellular origin in liver cancer Nat. Rev. Cancer 2015 15 653 667 10.1038/nrc4017 26493646 · DOI
  30. [35]Wang, D. A.-O., Tai, P. A.-O. & Gao, G. A.-O. Adeno-associated virus vector as a platform for gene therapy delivery. 18, 358–378 (2019). PMC6927556 30710128
  31. [36]Levy JM Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses Nat. Biomed. Eng. 2020 4 97 110 10.1038/s41551-019-0501-5 31937940 PMC6980783 · DOI
  32. [37]Zettler J Schütz V Mootz HD The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction FEBS Lett. 2009 583 909 914 10.1016/j.febslet.2009.02.003 19302791 · DOI
  33. [38]Wright AV Rational design of a split-Cas9 enzyme complex Proc. Natl Acad. Sci. USA 2015 112 2984 2989 10.1073/pnas.1501698112 25713377 PMC4364227 · DOI
  34. [39]Giannoukos G UDiTaS™, a genome editing detection method for indels and genome rearrangements BMC Genomics 2018 19 212 10.1186/s12864-018-4561-9 29562890 PMC5861650 · DOI
  35. [40]Yao X CRISPR/Cas9-mediated precise targeted integration in vivo using a double cut donor with short homology arms EBioMedicine 2017 20 19 26 10.1016/j.ebiom.2017.05.015 28527830 PMC5478232 · DOI
  36. [42]Xue W CRISPR-mediated direct mutation of cancer genes in the mouse liver Nature 2014 514 380 385 10.1038/nature13589 25119044 PMC4199937 · DOI · Paper page
  37. [45]Miura, K. Measurements of Intensity Dynamics at the Periphery of the Nucleus. (eds. Miura, K. & Sladoje, N) In: Bioimage Data Analysis Workflows. Learning Materials in Biosciences. (Springer, 2020).
  38. [47]Mane DR Kale AD Belaldavar C Validation of immunoexpression of tenascin-C in oral precancerous and cancerous tissues using ImageJ analysis with novel immunohistochemistry profiler plugin: an immunohistochemical quantitative analysis J. Oral Maxillofac. Pathol. 2017 21 211 217 10.4103/jomfp.JOMFP_234_16 28932029 PMC5596670 · DOI
  39. [48]Langmead B Salzberg SL Fast gapped-read alignment with Bowtie 2 Nat. Methods 2012 9 357 359 10.1038/nmeth.1923 22388286 PMC3322381 · DOI · Paper page
  40. [49]Li H The Sequence Alignment/Map format and SAMtools Bioinformatics 2009 25 2078 2079 10.1093/bioinformatics/btp352 19505943 PMC2723002 · DOI · Paper page
  41. [50]Ye K Schulz MH Long Q Apweiler R Ning Z Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads Bioinformatics 2009 25 2865 2871 10.1093/bioinformatics/btp394 19561018 PMC2781750 · DOI

Cited by

Sorted by citations

Authors
Liu, P., Liang, S.-Q., Zheng, C., Mintzer, E., Zhao, Y. G., Ponnienselvan, K., Mir, A., Sontheimer, E. J., Gao, G., Flotte, T. R., Wolfe, S. A., & Xue, W.
Affiliations
University of Massachusetts Chan Medical School (US)
Corresponding author
  • Wolfe, Scot A.
  • Xue, Wen
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-04-09
Volume
12
Issue
1
Pages
2121-2121
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-021-22295-w
PMC
PMC8035190
OpenAlex
W3145074783
Cited by
273
References
9
Citations by year
2026: 30 · 2025: 40 · 2024: 56 · 2023: 67 · 2022: 59 · 2021: 21
FWCI
16.08
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Cystic Fibrosis Foundation (UG3TR002668, R01GM115911)
  • Lung Cancer Research Foundation
  • Rett Syndrome Research Trust
  • National Institutes of Health (UG3TR002668, UH3TR002668)
  • National Heart, Lung, and Blood Institute (P01HL131471, DP2HL137167, UG3HL147367)
  • National Institute of General Medical Sciences (R01GM115911)
  • National Institute of Neurological Disorders and Stroke (R01NS111990)
  • National Center for Advancing Translational Sciences
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
prime editing, SpCas9, pegRNA, nuclear localization signal, adeno-associated virus vectors, tumor formation, cancer modelling, liver, adult mice, in vivo gene editing
MeSH terms
Gene Editing, Dependovirus, Alleles, Animals, Disease Models, Animal, HeLa Cells, Humans, Neoplasms, Transfection, Mice, HEK293 Cells, Carcinogenesis, CRISPR-Cas Systems

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review