Friday, April 24, 2026
No menu items!
HomeNatureQuadruple pegRNA enables programmable and efficient large genomic insertion

Quadruple pegRNA enables programmable and efficient large genomic insertion

  • Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Wang, J. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat. Methods 19, 331–340 (2022).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2023).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Pandey, S. et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nat. Biomed. Eng. 9, 22–39 (2025).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Witte, I. P. et al. Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase. Science 388, eadt5199 (2025).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • 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).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Sousa, A. A. et al. In vivo prime editing rescues alternating hemiplegia of childhood in mice. Cell 188, 4275–4294.e23 (2025).

  • Gillmore, J. D. et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 493–502 (2021).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • 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).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

    Article 
    CAS 
    PubMed 
    ADS 

    Google Scholar
     

  • Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Takata, M. et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17, 5497–5508 (1998).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Smirnikhina, S. A., Zaynitdinova, M. I., Sergeeva, V. A. & Lavrov, A. V. Improving homology-directed repair in genome editing experiments by influencing the cell cycle. Int. J. Mol. Sci. 23, 5992 (2022).

  • Hew, B. E. et al. Directed evolution of hyperactive integrases for site specific insertion of transgenes. Nucleic Acids Res. 52, e64 (2024).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Xu, Z. et al. Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol. 13, 87 (2013).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Romano, G., Marino, I. R., Pentimalli, F., Adamo, V. & Giordano, A. Insertional mutagenesis and development of malignancies induced by integrating gene delivery systems: implications for the design of safer gene-based interventions in patients. Drug News Perspect. 22, 185–196 (2009).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Zhang, R. et al. Amplification editing enables efficient and precise duplication of DNA from short sequence to megabase and chromosomal scale. Cell 187, 3936–3952 (2024).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002 (2023).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Yan, J. et al. Improving prime editing with an endogenous small RNA-binding protein. Nature 628, 639–647 (2024).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Lampe, G. D., Liang, A. R., Zhang, D. J., Fernández, I. S. & Sternberg, S. H. Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells. Nat. Commun. 16, 7891 (2025).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article 
    CAS 
    PubMed 
    ADS 

    Google Scholar
     

  • Park, J. et al. Digenome-seq web tool for profiling CRISPR specificity. Nat. Methods 14, 548–549 (2017).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Duan, M. et al. TOPO-seq reveals DNA topology-induced off-target activity by Cas9 and base editors. Nat. Chem. Biol. 21, 1554–1564 (2025).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Braal, C. L. et al. Inhibiting CDK4/6 in breast cancer with palbociclib, ribociclib, and abemaciclib: similarities and differences. Drugs 81, 317–331 (2021).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Crozier, L. et al. CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal. EMBO J. 41, e108599 (2022).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Wang, R. C. & Wang, Z. Synchronization of cultured cells to G1, S, G2, and M phases by double thymidine block. Methods Mol. Biol. 2579, 61–71 (2022).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Chen, G. & Deng, X. Cell synchronization by double thymidine block. Bio Protoc. 8, e2994 (2018).

  • Li, G., Yang, X., Luo, X., Wu, Z. & Yang, H. Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair. Cell Biosci. 13, 215 (2023).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Lei, X. et al. Rapid generation of long, chemically modified pegRNAs for prime editing. Nat. Biotechnol. 43, 1156–1167 (2025).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Cerbin, S. & Jiang, N. Duplication of host genes by transposable elements. Curr. Opin. Genet. Dev. 49, 63–69 (2018).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Skala, A. M. Retroviral DNA transposition: themes and variations. Microbiol. Spectr. 2, MDNA3-0005-2014 (2014).

  • Kahyo, T., Yamada, H., Tao, H., Kurabe, N. & Sugimura, H. Insertionally polymorphic sites of human endogenous retrovirus-K (HML-2) with long target site duplications. BMC Genom. 18, 487 (2017).

    Article 

    Google Scholar
     

  • Ewing, A. D. Transposable element detection from whole genome sequence data. Mob. DNA 6, 24 (2015).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Serrao, E., Ballandras-Colas, A., Cherepanov, P., Maertens, G. N. & Engelman, A. N. Key determinants of target DNA recognition by retroviral intasomes. Retrovirology 12, 39 (2015).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Sohrab, V., López-Díaz, C., Di Pietro, A., Ma, L. J. & Ayhan, D. H. TEfinder: a bioinformatics pipeline for detecting new transposable element insertion events in next-generation sequencing data. Genes 12, 224 (2021).

  • Devine, S. E., Chissoe, S. L., Eby, Y., Wilson, R. K. & Boeke, J. D. A transposon-based strategy for sequencing repetitive DNA in eukaryotic genomes. Genome Res. 7, 551–563 (1997).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Linheiro, R. S. & Bergman, C. M. Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster. PLoS ONE 7, e30008 (2012).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Yao, X. et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 27, 801–814 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Shy, B. R. et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat. Biotechnol. 41, 521–531 (2023).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Miyaoka, Y. et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci. Rep. 6, 23549 (2016).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Iyama, T. & Wilson, D. M. III DNA repair mechanisms in dividing and non-dividing cells. DNA Repair 12, 620–636 (2013).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Frock, R. L., Sadeghi, C., Meng, J. & Wang, J. L. DNA end joining: G0-ing to the core. Biomolecules 11, 1487 (2021).

  • Yu, W. et al. Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ. Nat. Commun. 11, 5239 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • An, J. et al. Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway. Nat. Biomed. Eng. 8, 149–164 (2024).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Zhang, C. P. et al. Efficient non-viral delivery of macromolecules in human primary hematopoietic stem cells and lymphocytes. J. Mol. Cell. Biol. 15, mjad018 (2023).

  • Zhang, A. et al. Efficient and precise inversion of genomic DNA from large to chromosomal scale. Nat. Chem. Biol. 22, 328–339 (2025).

  • Ji, R. J. et al. Epitope prime editing shields hematopoietic cells from CD123 immunotherapy for acute myeloid leukemia. Cell Stem Cell 31, 1650–1666 (2024).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Wang, G. et al. mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation. Cell Insight 1, 100056 (2022).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Tong, X. et al. Structure-guided design of Cas12a variants improves detection of nucleic acids. Cell Insight 4, 100228 (2025).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 
    ADS 

    Google Scholar
     

  • Shi, Y. J. et al. DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9. Mol. Cell 82, 4160–4175 (2022).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Kim, D., Kang, B. C. & Kim, J. S. Identifying genome-wide off-target sites of CRISPR RNA-guided nucleases and deaminases with Digenome-seq. Nat. Protoc. 16, 1170–1192 (2021).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Su, X. et al. The promising role of nanopore sequencing in cancer diagnostics and treatment. Cell Insight 4, 100229 (2025).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Robinson, J. T. et al. Integrative Genomics Viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • RELATED ARTICLES

    Most Popular

    Recent Comments