ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models

abe9-fused-to-spry-cas9-nickase-enables-precise-generation-of-bystander-free-mouse-models
ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models
  • Williams, R. W. in Principles of Molecular Medicine (eds Runge, M. S. & Patterson, C.) 53–60 (Humana Press, 2006). https://doi.org/10.1007/978-1-59259-963-9_8

  • Perlman, R. L. Mouse models of human disease: An evolutionary perspective. Evol. Med. Public Health 2016, 170–176. https://doi.org/10.1093/emph/eow014 (2016).

    Google Scholar 

  • Smith, P., DiLillo, D. J., Bournazos, S., Li, F. & Ravetch, J. V. Mouse model recapitulating human Fcγ receptor structural and functional diversity. Proc. Natl. Acad. Sci. U. S. A. 109, 6181–6186. https://doi.org/10.1073/pnas.1203954109 (2012).

    Google Scholar 

  • Scherrer, G. et al. Knockin mice expressing fluorescent δ-opioid receptors uncover G protein-coupled receptor dynamics in vivo. Proc. Natl. Acad. Sci. U. S. A. 103, 9691–9696. https://doi.org/10.1073/pnas.0603359103 (2006).

    Google Scholar 

  • Lee, H. Genetically engineered mouse models for drug development and preclinical trials. Biomol. Ther. 22, 267–274. https://doi.org/10.4062/biomolther.2014.074 (2014).

    Google Scholar 

  • Passier, R., Orlova, V. & Mummery, C. Complex tissue and disease modeling using hiPSCs. Cell Stem Cell 18, 309–321. https://doi.org/10.1016/j.stem.2016.02.011 (2016).

    Google Scholar 

  • Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. https://doi.org/10.1126/science.1231143 (2013).

    Google Scholar 

  • Chang, H. H. Y., Pannunzio, N. R., Adachi, N. & Lieber, M. R. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat. Rev. Mol. Cell Biol. 18, 495–506. https://doi.org/10.1038/nrm.2017.48 (2017).

    Google Scholar 

  • Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826. https://doi.org/10.1038/nbt.2623 (2013).

    Google Scholar 

  • Hwang, G.-H. et al. Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases. Nat. Biomed. Eng. 9, 79–92. https://doi.org/10.1038/s41551-024-01277-5 (2025).

    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. https://doi.org/10.1038/nbt.4192 (2018).

    Google Scholar 

  • Cullot, G. et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat. Commun. 10, 1136. https://doi.org/10.1038/s41467-019-09006-2 (2019).

    Google Scholar 

  • Weisheit, I. et al. Detection of deleterious on-target effects after HDR-mediated CRISPR editing. Cell Rep. 31, 107689. https://doi.org/10.1016/j.celrep.2020.107689 (2020).

    Google Scholar 

  • Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 53, 895–905. https://doi.org/10.1038/s41588-021-00838-7 (2021).

    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. https://doi.org/10.1038/nature24644 (2017).

    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. https://doi.org/10.1038/nature17946 (2016).

    Google Scholar 

  • Rees, H. A. & Liu, D. R. Base editing: Precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788. https://doi.org/10.1038/s41576-018-0059-1 (2018).

    Google Scholar 

  • Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900. https://doi.org/10.1038/s41587-020-0491-6 (2020).

    Google Scholar 

  • Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676. https://doi.org/10.1038/nbt.2889 (2014).

    Google Scholar 

  • Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832. https://doi.org/10.1038/nbt.2647 (2013).

    Google Scholar 

  • Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437. https://doi.org/10.1038/s41586-019-1161-z (2019).

    Google Scholar 

  • Li, S., Liu, L., Sun, W., Zhou, X. & Zhou, H. A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants. Genome Biol. 23, 51. https://doi.org/10.1186/s13059-022-02618-w (2022).

    Google Scholar 

  • Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891. https://doi.org/10.1038/s41587-020-0453-z (2020).

    Google Scholar 

  • Chen, L. et al. Engineering a precise adenine base editor with minimal bystander editing. Nat. Chem. Biol. 19, 101–110. https://doi.org/10.1038/s41589-022-01163-8 (2023).

    Google Scholar 

  • Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290–296. https://doi.org/10.1126/science.aba8853 (2020).

    Google Scholar 

  • Liao, J. et al. Therapeutic adenine base editing of human hematopoietic stem cells. Nat. Commun. 14, 207. https://doi.org/10.1038/s41467-022-35508-7 (2023).

    Google Scholar 

  • Patel, S. & Kilpatrick, B. S. Two-pore channels and disease. Biochim. Biophys. Acta (BBA) Mol. Cell Res. 1865, 1678–1686. https://doi.org/10.1016/j.bbamcr.2018.05.004 (2018).

    Google Scholar 

  • García-Rúa, V. et al. Increased expression of fatty-acid and calcium metabolism genes in failing human heart. PLoS ONE 7, e37505. https://doi.org/10.1371/journal.pone.0037505 (2012).

    Google Scholar 

  • Deutsch, R., Kudrina, V., Freichel, M. & Grimm, C. Two-pore channel regulators—Who is in control?. Front. Physiol. https://doi.org/10.3389/fphys.2024.1534071 (2025).

    Google Scholar 

  • Medert, R. et al. Genetic background influences expression and function of the cation channel TRPM4 in the mouse heart. Basic Res. Cardiol. 115, 70. https://doi.org/10.1007/s00395-020-00831-x (2020).

    Google Scholar 

  • Stallmeyer, B. et al. Mutational spectrum in the Ca2+-activated cation channel gene TRPM4 in patients with cardiac conductance disturbances. Hum. Mutat. 33, 109–117. https://doi.org/10.1002/humu.21599 (2012).

    Google Scholar 

  • Tu, T. et al. A precise and efficient adenine base editor. Mol. Ther. 30, 2933–2941. https://doi.org/10.1016/j.ymthe.2022.07.010 (2022).

    Google Scholar 

  • Jeong, Y. K. et al. Adenine base editor engineering reduces editing of bystander cytosines. Nat. Biotechnol. 39, 1426–1433. https://doi.org/10.1038/s41587-021-00943-2 (2021).

    Google Scholar 

  • Cao, X. et al. Engineering of near-PAMless adenine base editor with enhanced editing activity and reduced off-target. Mol. Ther. Nucleic Acids 28, 732–742. https://doi.org/10.1016/j.omtn.2022.04.032 (2022).

    Google Scholar 

  • Zhang, Z. et al. Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities. Mol. Ther. Nucleic Acids 29, 502–510. https://doi.org/10.1016/j.omtn.2022.07.026 (2022).

    Google Scholar 

  • Li, G. et al. A novel base editor SpRY-ABE8eF148A mediates efficient A-to-G base editing with a reduced off-target effect. Mol. Ther. Nucleic Acids 31, 78–87. https://doi.org/10.1016/j.omtn.2022.12.001 (2023).

    Google Scholar 

  • Xiong, X. et al. Split complementation of base editors to minimize off-target edits. Nat. Plants 9, 1832–1847. https://doi.org/10.1038/s41477-023-01540-8 (2023).

    Google Scholar 

  • Zeng, H. et al. A split and inducible adenine base editor for precise in vivo base editing. Nat. Commun. 14, 5573. https://doi.org/10.1038/s41467-023-41331-5 (2023).

    Google Scholar 

  • Kim, Y.-h et al. Sniper2L is a high-fidelity Cas9 variant with high activity. Nat. Chem. Biol. 19, 972–980. https://doi.org/10.1038/s41589-023-01279-5 (2023).

    Google Scholar 

  • Wang, Z. et al. Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant. Int. J. Biol. Macromol. 253, 127418. https://doi.org/10.1016/j.ijbiomac.2023.127418 (2023).

    Google Scholar 

  • Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63. https://doi.org/10.1038/nature26155 (2018).

    Google Scholar 

  • Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485. https://doi.org/10.1038/nature14592 (2015).

    Google Scholar 

  • Huang, T. P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat. Biotechnol. 37, 626–631. https://doi.org/10.1038/s41587-019-0134-y (2019).

    Google Scholar 

  • Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262. https://doi.org/10.1126/science.aas9129 (2018).

    Google Scholar 

  • Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191. https://doi.org/10.1038/nature14299 (2015).

    Google Scholar 

  • Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481. https://doi.org/10.1038/s41587-020-0412-8 (2020).

    Google Scholar 

  • Zhao, L. et al. PAM-flexible genome editing with an engineered chimeric Cas9. Nat. Commun. 14, 6175. https://doi.org/10.1038/s41467-023-41829-y (2023).

    Google Scholar 

  • Li, J. et al. Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants. Mol. Plant 14, 352–360. https://doi.org/10.1016/j.molp.2020.12.017 (2021).

    Google Scholar 

  • Villiger, L. et al. In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA. Nat. Biomed. Eng. 5, 179–189. https://doi.org/10.1038/s41551-020-00671-z (2021).

    Google Scholar 

  • Liu, Z. et al. Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing. Nat. Commun. 9, 2338. https://doi.org/10.1038/s41467-018-04768-7 (2018).

    Google Scholar 

  • Ryu, S.-M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539. https://doi.org/10.1038/nbt.4148 (2018).

    Google Scholar 

  • Yang, L. et al. Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 9, 814–819. https://doi.org/10.1007/s13238-018-0568-x (2018).

    Google Scholar 

  • Fu, J. et al. Human cell based directed evolution of adenine base editors with improved efficiency. Nat. Commun. 12, 5897. https://doi.org/10.1038/s41467-021-26211-0 (2021).

    Google Scholar 

  • Qian, Y. et al. A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9. BMC Biol. 21, 155. https://doi.org/10.1186/s12915-023-01644-9 (2023).

    Google Scholar 

  • Zhao, D. et al. Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope. BMC Biol. 21, 250. https://doi.org/10.1186/s12915-023-01754-4 (2023).

    Google Scholar 

  • Cornean, A. et al. Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction. Elife 11, e72124. https://doi.org/10.7554/eLife.72124 (2022).

    Google Scholar 

  • Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. R. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. 38, 620–628. https://doi.org/10.1038/s41587-020-0414-6 (2020).

    Google Scholar 

  • Joshi, J., Albers, C., Smole, N., Guo, S. & Smith, S. A. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling cardiac arrhythmias: Strengths, challenges and potential solutions. Front. Physiol. https://doi.org/10.3389/fphys.2024.1475152 (2024).

    Google Scholar 

  • Cerneckis, J., Cai, H. & Shi, Y. Induced pluripotent stem cells (iPSCs): Molecular mechanisms of induction and applications. Signal Transduct. Target. Ther. 9, 112. https://doi.org/10.1038/s41392-024-01809-0 (2024).

    Google Scholar 

  • Brookhouser, N. et al. A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells. BMC Biol. 18, 193. https://doi.org/10.1186/s12915-020-00929-7 (2020).

    Google Scholar 

  • Rosello, M. et al. Disease modeling by efficient genome editing using a near PAM-less base editor in vivo. Nat. Commun. 13, 3435. https://doi.org/10.1038/s41467-022-31172-z (2022).

    Google Scholar 

  • Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292. https://doi.org/10.1126/science.aav9973 (2019).

    Google Scholar 

  • Lee, S.-H. et al. Bystander editing by adenine base editors impairs vision restoration in a mouse model of Leber congenital amaurosis. Mol. Ther. Methods Clin. Dev. https://doi.org/10.1016/j.omtm.2025.101461 (2025).

    Google Scholar 

  • Tuladhar, R. et al. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat. Commun. 10, 4056. https://doi.org/10.1038/s41467-019-12028-5 (2019).

    Google Scholar 

  • Höijer, I. et al. CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nat. Commun. 13, 627. https://doi.org/10.1038/s41467-022-28244-5 (2022).

    Google Scholar 

  • Perrotta, R. M. et al. Engineered base editors with reduced bystander editing through directed evolution. Nat. Biotechnol. https://doi.org/10.1038/s41587-025-02937-w (2025).

    Google Scholar 

  • Xiao, Y.-L., Wu, Y. & Tang, W. An adenine base editor variant expands context compatibility. Nat. Biotechnol. 42, 1442–1453. https://doi.org/10.1038/s41587-023-01994-3 (2024).

    Google Scholar 

  • Qin, W. et al. ABE-ultramax for high-efficiency biallelic adenine base editing in zebrafish. Nat. Commun. 15, 5613. https://doi.org/10.1038/s41467-024-49943-1 (2024).

    Google Scholar 

  • Zhao, N. et al. Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells. Nat. Commun. 15, 8140. https://doi.org/10.1038/s41467-024-52483-3 (2024).

    Google Scholar 

  • Chen, Q. et al. Engineering of peptide-inserted base editors with enhanced accuracy and security. Small 21, 2411583. https://doi.org/10.1002/smll.202411583 (2025).

    Google Scholar 

  • Liao, J. et al. Sequential amino acid mutagenesis-driven de novo evolution of adenine deaminases enables efficient in vivo base editing in primate. bioRxiv. 2025.2005.2014.653640 (2025). https://doi.org/10.1101/2025.05.14.653640

  • Silverstein, R. A. et al. Custom CRISPR–Cas9 PAM variants via scalable engineering and machine learning. Nature https://doi.org/10.1038/s41586-025-09021-y (2025).

    Google Scholar 

  • Fiumara, M. et al. Genotoxic effects of base and prime editing in human hematopoietic stem cells. Nat. Biotechnol. 42, 877–891. https://doi.org/10.1038/s41587-023-01915-4 (2024).

    Google Scholar 

  • Peña-Gutiérrez, I., Olalla-Sastre, B., Río, P. & Rodríguez-Madoz, J. R. Beyond precision: Evaluation of off-target clustered regularly interspaced short palindromic repeats/Cas9-mediated genome editing. Cytotherapy 27, 279–286. https://doi.org/10.1016/j.jcyt.2024.10.010 (2025).

    Google Scholar 

  • Liang, P. et al. Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat. Commun. 10, 67. https://doi.org/10.1038/s41467-018-07988-z (2019).

    Google Scholar 

  • Kim, D., Kim, D.-e, Lee, G., Cho, S.-I. & Kim, J.-S. Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat. Biotechnol. 37, 430–435. https://doi.org/10.1038/s41587-019-0050-1 (2019).

    Google Scholar 

  • Yuan, K. et al. Selict-seq profiles genome-wide off-target effects in adenosine base editing. Nucleic Acids Res. https://doi.org/10.1093/nar/gkaf281 (2025).

    Google Scholar 

  • Turchiano, G. et al. Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq. Cell Stem Cell 28, 1136-1147.e1135. https://doi.org/10.1016/j.stem.2021.02.002 (2021).

    Google Scholar 

  • Shi, Y., Inoue, H., Wu, J. C. & Yamanaka, S. Induced pluripotent stem cell technology: A decade of progress. Nat. Rev. Drug Discov. 16, 115–130. https://doi.org/10.1038/nrd.2016.245 (2017).

    Google Scholar 

  • Heinzelmann, E. et al. iPSC-derived and patient-derived organoids: Applications and challenges in scalability and reproducibility as pre-clinical models. Curr. Res. Toxicol. 7, 100197. https://doi.org/10.1016/j.crtox.2024.100197 (2024).

    Google Scholar 

  • Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157. https://doi.org/10.1038/s41586-019-1711-4 (2019).

    Google Scholar 

  • Alves, C. R. R. et al. Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy. Nat. Biomed. Eng. 8, 118–131. https://doi.org/10.1038/s41551-023-01132-z (2024).

    Google Scholar 

  • Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635-5652.e5629. https://doi.org/10.1016/j.cell.2021.09.018 (2021).

    Google Scholar 

  • Medert, R. et al. Efficient single copy integration via homology-directed repair (scHDR) by 5′modification of large DNA donor fragments in mice. Nucleic Acids Res. 51, e14–e14. https://doi.org/10.1093/nar/gkac1150 (2022).

    Google Scholar 

  • Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226. https://doi.org/10.1038/s41587-019-0032-3 (2019).

    Google Scholar 

  • Doman, J. L., Sousa, A. A., Randolph, P. B., Chen, P. J. & Liu, D. R. Designing and executing prime editing experiments in mammalian cells. Nat. Protoc. 17, 2431–2468. https://doi.org/10.1038/s41596-022-00724-4 (2022).

    Google Scholar 

  • Wickham, H. et al. Welcome to the tidyverse. J. Open Source Softw. 4, 1686. https://doi.org/10.21105/joss.01686 (2019).

    Google Scholar 

  • Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, 2016). https://doi.org/10.18637/jss.v035.b01.

    Google Scholar 

  • Kassambara, A. ggpubr: ‘ggplot2’ Based Publication Ready Plots (2022). https://doi.org/10.32614/CRAN.package.ggpubr

  • Wilke, C. O. cowplot: Streamlined Plot Theme and Plot Annotations for ‘ggplot2’. (2024). https://doi.org/10.32614/CRAN.package.cowplot