CoChat
Sign Up
Cited by 613CRISPR and Genetic EngineeringRNA regulation and diseaseDNA Repair Mechanisms

Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing

Open access
Anzalone, Andrew V., Gao, Xin D., Podracky, Christopher J., Nelson, Andrew T., Koblan, Luke W., Raguram, Aditya, Levy, Jonathan M., Mercer, Jaron A. M. +1 more·2022·Nature Biotechnology
DOI 10.1038/s41587-021-01133-wPMC PMC9117393
Open this paper in CoChat — free account

Abstract

The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.

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/s41587-021-01133-w

Cites

11 of 11 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?

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: 48

  1. [2]Landrum MJ ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res 42, D980– 985, doi: 10.1093/nar/gkt1113 (2014). 24234437 PMC3965032 · DOI · Paper page
  2. [3]Weischenfeldt J, Symmons O, Spitz F & Korbel JO Phenotypic impact of genomic structural variation: insights from and for human disease. Nat Rev Genet 14, 125– 138, doi: 10.1038/nrg3373 (2013). 23329113 · DOI
  3. [4]Cox DB, Platt RJ & Zhang F Therapeutic genome editing: prospects and challenges. Nat Med 21, 121– 131, doi: 10.1038/nm.3793 (2015). 25654603 PMC4492683 · DOI · Paper page
  4. [6]Anzalone AV, Koblan LW & Liu DR Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 38, 824– 844, doi: 10.1038/s41587-020-0561-9 (2020). 32572269 · DOI · Paper page
  5. [9]Mali P RNA-guided human genome engineering via Cas9. Science 339, 823– 826, doi: 10.1126/science.1232033 (2013). 23287722 PMC3712628 · DOI · Paper page
  6. [12]Anzalone AV Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149– 157, doi: 10.1038/s41586-019-1711-4 (2019). 31634902 PMC6907074 · DOI · Paper page
  7. [13]Canver MC Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. The Journal of biological chemistry 289, 21312– 21324, doi: 10.1074/jbc.M114.564625 (2014). 24907273 PMC4118095 · DOI
  8. [14]Suzuki K In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144– 149, doi: 10.1038/nature20565 (2016). 27851729 PMC5331785 · DOI · Paper page
  9. [15]Wang B Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes. Biotechniques 59, 201– 202, 204, 206-208, doi: 10.2144/000114339 (2015). 26458548 · DOI
  10. [16]Pawelczak KS, Gavande NS, VanderVere-Carozza PS & Turchi JJ Modulating DNA Repair Pathways to Improve Precision Genome Engineering. ACS Chem Biol 13, 389– 396, doi: 10.1021/acschembio.7b00777 (2018). 29210569 · DOI
  11. [20]Kosicki M, Tomberg K & Bradley A Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nature Biotechnology 36, 765– 771, doi: 10.1038/nbt.4192 (2018). PMC6390938 30010673 · DOI · Paper page
  12. [22]Song Y Large-Fragment Deletions Induced by Cas9 Cleavage while Not in the BEs System. Molecular Therapy - Nucleic Acids 21, 523– 526, doi: 10.1016/j.omtn.2020.06.019 (2020). 32711379 PMC7381496 · DOI
  13. [23]Brunet E & Jasin M Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations. Advances in experimental medicine and biology 1044, 15– 25, doi: 10.1007/978-981-13-0593-1_2 (2018). 29956288 PMC6333474 · DOI
  14. [24]Nahmad AD Frequent Aneuploidy in Primary Human T Cells Following CRISPR-Cas9 cleavage. bioRxiv, 2021.2008.2020.457092, doi: 10.1101/2021.08.20.457092 (2021). · DOI
  15. [25]Leibowitz ML Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nature Genetics, doi: 10.1038/s41588-021-00838-7 (2021). PMC8192433 33846636 · DOI · Paper page
  16. [26]Haapaniemi E, Botla S, Persson J, Schmierer B & Taipale J CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nature Medicine 24, 927– 930, doi: 10.1038/s41591-018-0049-z (2018). 29892067 · DOI · Paper page
  17. [27]Ihry RJ p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nature Medicine 24, 939– 946, doi: 10.1038/s41591-018-0050-6 (2018). 29892062 · DOI · Paper page
  18. [28]Enache OM Cas9 activates the p53 pathway and selects for p53-inactivating mutations. Nat Genet 52, 662– 668, doi: 10.1038/s41588-020-0623-4 (2020). 32424350 PMC7343612 · DOI · Paper page
  19. [29]Merrick CA, Zhao J & Rosser SJ Serine Integrases: Advancing Synthetic Biology. ACS Synthetic Biology 7, 299– 310, doi: 10.1021/acssynbio.7b00308 (2018). 29316791 · DOI
  20. [30]Karpinski J Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotechnol 34, 401– 409, doi: 10.1038/nbt.3467 (2016). 26900663 · DOI
  21. [31]Chaikind B, Bessen JL, Thompson DB, Hu JH & Liu DR A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Res 44, 9758– 9770, doi: 10.1093/nar/gkw707 (2016). 27515511 PMC5175349 · DOI
  22. [32]Gaj T Enhancing the Specificity of Recombinase-Mediated Genome Engineering through Dimer Interface Redesign. J Am Chem Soc 136, 5047– 5056, doi: 10.1021/ja4130059 (2014). 24611715 PMC3985937 · DOI
  23. [33]Kim AI Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Molecular Microbiology 50, 463– 473, doi: 10.1046/j.1365-2958.2003.03723.x (2003). 14617171 · DOI
  24. [34]Choi J Precise genomic deletions using paired prime editing. Nat Biotechnol, doi: 10.1038/s41587-021-01025-z (2021). PMC8847327 34650269 · DOI
  25. [35]Lin Q High-efficiency prime editing with optimized, paired pegRNAs in plants. Nature Biotechnology, doi: 10.1038/s41587-021-00868-w (2021). 33767395 · DOI · Paper page
  26. [36]Scriver CR The PAH gene, phenylketonuria, and a paradigm shift. Human Mutation 28, 831– 845, doi: 10.1002/humu.20526 (2007). 17443661 · DOI
  27. [37]Nelson JW Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol, doi: 10.1038/s41587-021-01039-7 (2021). PMC8930418 34608327 · DOI · Paper page
  28. [38]Flanigan KM Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Human mutation 30, 1657– 1666, doi: 10.1002/humu.21114 (2009). 19937601 PMC3404892 · DOI
  29. [39]Aartsma-Rus A Development of Exon Skipping Therapies for Duchenne Muscular Dystrophy: A Critical Review and a Perspective on the Outstanding Issues. Nucleic acid therapeutics 27, 251– 259, doi: 10.1089/nat.2017.0682 (2017). 28796573 PMC5649120 · DOI
  30. [40]Kim DY, Moon SB, Ko J-H, Kim Y-S & Kim D Unbiased investigation of specificities of prime editing systems in human cells. Nucleic Acids Research 48, 10576– 10589, doi: 10.1093/nar/gkaa764 (2020). 32941652 PMC7544197 · DOI
  31. [41]Jin S Genome-wide specificity of prime editors in plants. Nature Biotechnology, doi: 10.1038/s41587-021-00891-x (2021). 33859403 · DOI
  32. [42]Duportet X A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic acids research 42, 13440– 13451, doi: 10.1093/nar/gku1082 (2014) 25378321 PMC4245948 · DOI
  33. [43]Jusiak B Comparison of Integrases Identifies Bxb1-GA Mutant as the Most Efficient Site-Specific Integrase System in Mammalian Cells. ACS Synthetic Biology 8, 16– 24, doi: 10.1021/acssynbio.8b00089 (2019). 30609349 · DOI
  34. [44]Sharma R In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood 126, 1777– 1784, doi: 10.1182/blood-2014-12-615492 PMC4600017 26297739 · DOI · Paper page
  35. [46]Bessen JL High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases. Nature Communications 10, 1937, doi: 10.1038/s41467-019-09987-0 (2019). PMC6486577 31028261 · DOI
  36. [47]Bondeson ML Inversion of the IDS gene resulting from recombination with IDS-related sequences is a common cause of the Hunter syndrome. Human molecular genetics 4, 615– 621, doi: 10.1093/hmg/4.4.615 (1995). 7633410 · DOI
  37. [48]Chen X In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting. Nat Commun 8, 657, doi: 10.1038/s41467-017-00687-1 (2017). 28939824 PMC5610252 · DOI
  38. [49]Park CY Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs. Proc Natl Acad Sci U S A 111, 9253– 9258, doi: 10.1073/pnas.1323941111 (2014). 24927536 PMC4078797 · DOI
  39. [50]Li J Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9. Journal of molecular cell biology 7, 284– 298, doi: 10.1093/jmcb/mjv016 (2015). 25757625 PMC4524425 · DOI
  40. [1]Anzalone AV Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149– 157, doi: 10.1038/s41586-019-1711-4 (2019). 31634902 PMC6907074 · DOI · Paper page
  41. [2]Komor AC, Kim YB, Packer MS, Zuris JA & Liu DR Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420– 424, doi: 10.1038/nature17946 (2016). 27096365 PMC4873371 · DOI · Paper page
  42. [3]Clement K CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nature Biotechnology 37, 224– 226, doi: 10.1038/s41587-019-0032-3 (2019). PMC6533916 30809026 · DOI
  43. [4]Clement K, Farouni R, Bauer DE & Pinello L AmpUMI: design and analysis of unique molecular identifiers for deep amplicon sequencing. Bioinformatics 34, i202– i210, doi: 10.1093/bioinformatics/bty264 (2018). 29949956 PMC6022702 · DOI
  44. [5]Shen W, Le S, Li Y & Hu F SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation. PLoS One 11, e0163962, doi: 10.1371/journal.pone.0163962 (2016). 27706213 PMC5051824 · DOI · Paper page
  45. [6]Levy JM & Nicoll RA Membrane-associated guanylate kinase dynamics reveal regional and developmental specificity of synapse stability. The Journal of Physiology 595, 1699– 1709, doi: 10.1113/JP273147 (2017). 27861918 PMC5330878 · DOI
  46. [7]Koblan LW In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice. Nature 589, 608– 614, doi: 10.1038/s41586-020-03086-7 (2021). 33408413 PMC7872200 · DOI
  47. [8]Gaudelli NM Directed evolution of adenine base editors with increased activity and therapeutic application. Nature Biotechnology 38, 892– 900, doi: 10.1038/s41587-020-0491-6 (2020). 32284586 · DOI
  48. [9]Nelson JW Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol, doi: 10.1038/s41587-021-01039-7 (2021). PMC8930418 34608327 · DOI · Paper page

Cited by

Sorted by citations

Authors
Anzalone, A. V., Gao, X. D., Podracky, C. J., Nelson, A. T., Koblan, L. W., Raguram, A., Levy, J. M., Mercer, J. A. M., & Liu, D. R.
Affiliations
Broad Institute (US)
Corresponding author
Liu, David R.
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2021-12-09
Volume
40
Issue
5
Pages
731-740
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/s41587-021-01133-w
PMC
PMC9117393
OpenAlex
W4200345742
Cited by
613
References
11
Citations by year
2026: 129 · 2025: 143 · 2024: 141 · 2023: 128 · 2022: 71 · 2021: 1
FWCI
26.84
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
Funders
  • U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (RM1 HG00949)
  • U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (U01 AI142756)
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35 GM118062)
  • Howard Hughes Medical Institute (Liu investigatorship)
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 › Molecular Biology › DNA Repair Mechanisms
Keywords
prime editing, prime editors, pegRNA, serine recombinase
MeSH terms
Gene Editing, RNA, Guide, CRISPR-Cas Systems, Base Sequence, DNA, Humans, Chromosome Inversion, RNA, Guide, Kinetoplastida, 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