Optimized base editors enable efficient editing in cells, organoids and mice
Open accessAbstract
CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.
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.
Integrity signals
Registry
Cites
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.
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?
The described protocols are applicable in ASC-derived organoids from both murine and human sources and can be derived from various organs.
“These protocols are applicable in ASC derived organoids from both murine and human sources and can be derived from various organs.17”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [17]
The Online Methods section on organoid isolation and transfection describes protocols for murine small-intestinal organoids. It does not establish their applicability to human ASC-derived organoids or organoids from various organs. The paper’s mention of human organoids describes work reported elsewhere, not an application of these protocols. (LLM, conf=0.86)
Experimental — accuracy not yet measuredCBEs have been utilized to mutate the S45 residue, effectively modeling the first step of the phosphorylation cascade.
“Previously, Zafra and colleagues have utilized CBEs to mutate the S45 residue, effectively modeling the first step of the phosphorylation cascade36.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [36]
The paper reports using base editors to create CTNNB1 S45F mutations in DLD1 cells and mouse liver (main text, Figure 3a–b and 3f–g). It describes the mutations as activating WNT signaling, but does not say that they model the first step of a phosphorylation cascade. (LLM, conf=0.85)
Experimental — accuracy not yet measuredBase editors have been applied before in organoids to model and repair diseases, and this study significantly extends their application by demonstrating opportunities in multiplexing CBEs and ABEs, Cas9 homologs, and evolved Cas9 variants.
“While base editors have been applied before in organoids to model36 and repair31 diseases, we significantly extend their application by demonstrating the opportunities that reside in multiplexing CBE’s and ABE’s, as well as Cas9 homologs and evolved Cas9 variants.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [36]
CTNNB1 was mainly localized at the plasma membrane in wild-type organoids, whereas intracellular levels in the cytoplasm and nucleus was low.
“As expected, CTNNB1 was mainly localized at the plasma membrane in wild-type organoids, presumably bound to adherens junctions36, whereas intracellular levels in the cytoplasm and nucleus was low (Fig. 2d, Supplementary Fig. 3b).”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [36]
The citation is attached to the suggestion that membrane-localized CTNNB1 is bound to adherens junctions. The paper’s organoid passages describe base editing and culture methods, not CTNNB1 localization; searches for plasma-membrane localization and adherens junctions found no passage addressing that point. The paper also does not report the claimed low cytoplasmic and nuclear levels in wild-type organoids. (LLM, conf=0.73)
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
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
Comprehensive molecular characterization of human colon and rectal cancer
The Cancer Genome Atlas Network (2012) · Nature · doi:10.1038/nature11252 · Cited by 8,499 · see citing works
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016) · Nature · doi:10.1038/nature17946 · Cited by 5,437 · see citing works
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017) · Nature · doi:10.1038/nature24644 · Cited by 4,184 · see citing works
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3117 · Cited by 2,268 · see citing works
Engineered CRISPR-Cas9 nucleases with altered PAM specificities
Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., Gonzales, A. P. W., Li, Z., Peterson, R. T., Yeh, J.-R. J., et al. (2015) · Nature · doi:10.1038/nature14592 · Cited by 1,624 · see citing works
Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems
Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K. Y., et al. (2016) · Science · doi:10.1126/science.aaf8729 · Cited by 1,349 · see citing works
Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing
Yin, H., Song, C.-Q., Suresh, S., Wu, Q., Walsh, S., Rhym, L. H., Mintzer, E., Bolukbasi, M. F., Zhu, L. J., Kauffman, K., et al. (2017) · Nature Biotechnology · doi:10.1038/nbt.4005 · Cited by 499 · see citing works
Flagged first, then by citations
Research works printed but not linked: 24
- [3]Hess GT et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat Methods 13, 1036– 1042 (2016). 27798611 PMC5557288
- [4]Ma Y et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat Methods 13, 1029– 1035 (2016). 27723754
- [6]Cox DBT et al. RNA editing with CRISPR-Cas13. Science 358, 1019– 1027 (2017). 29070703 PMC5793859
- [7]Rees HA et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun 8, 15790 (2017). 28585549 PMC5467206
- [8]Kim K et al. Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol 35, 435– 437 (2017). 28244995
- [10]Kim S, Bae T, Hwang J & Kim JS Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides. Genome Biol 18, 218 (2017). 29141659 PMC5686910
- [11]Kleinstiver BP et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490– 495 (2016). 26735016 PMC4851738
- [13]Dow LE et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol 33, 390– 394 (2015). 25690852 PMC4390466
- [14]Staahl BT et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol 35, 431– 434 (2017). 28191903 PMC6649674
- [15]Komor AC et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv 3, eaao4774 (2017). 28875174 PMC5576876
- [16]Huang SM et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614– 620 (2009). 19759537
- [17]Schoumacher M et al. Inhibiting Tankyrases sensitizes KRAS-mutant cancer cells to MEK inhibitors via FGFR2 feedback signaling. Cancer Res 74, 3294– 3305 (2014). 24747911
- [18]Mashima T et al. mTOR signaling mediates resistance to tankyrase inhibitors in Wnt-driven colorectal cancer. Oncotarget 8, 47902– 47915 (2017). 28615517 PMC5564614
- [20]Matano M et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nature medicine 21, 256– 262 (2015). 25706875
- [21]Cadoret A et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 21, 8293– 8301 (2002). 12447692
- [22]Annunziato S et al. Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland. Genes Dev 30, 1470– 1480 (2016). 27340177 PMC4926868
- [23]Wang D et al. Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses. Human gene therapy 26, 432– 442 (2015). 26086867 PMC4509492
- [24]Hu JH et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57– 63 (2018). 29512652 PMC5951633
- [25]Koblan LW et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol (2018). PMC6126947 29813047
- [26]Ryu SM et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat Biotechnol 36, 536– 539 (2018). 29702637
- [28]Dow LE et al. A pipeline for the generation of shRNA transgenic mice. Nature protocols (2012). PMC3724521 22301776
- [29]Han T et al. R-Spondin chromosome rearrangements drive Wnt-dependent tumour initiation and maintenance in the intestine. Nat Commun 8, 15945 (2017). 28695896 PMC5508203
- [30]O’Rourke KP et al. Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer. Nat Biotechnol 35, 577– 582 (2017). 28459450 PMC5462850
- [32]Bae S, Park J & Kim JS Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473– 1475 (2014). 24463181 PMC4016707
Cited by
Sorted by citations
Base editing: precision chemistry on the genome and transcriptome of living cells
Rees, H. A., & Liu, D. R. (2018) · Nature Reviews Genetics · doi:10.1038/s41576-018-0059-1 · Cited by 1,675 · see citing works
Richter, M. F., Zhao, K. T., Eton, E., Lapinaite, A., Newby, G. A., Thuronyi, B. W., Wilson, C., Koblan, L. W., Zeng, J., Bauer, D. E., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0453-z · Cited by 1,020 · see citing works
Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis
Zhou, C., Sun, Y., Yan, R., Liu, Y., Zuo, E., Gu, C., Han, L., Wei, Y., Hu, X., Zeng, R., et al. (2019) · Nature · doi:10.1038/s41586-019-1314-0 · Cited by 482 · see citing works
Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors
Doman, J. L., Raguram, A., Newby, G. A., & Liu, D. R. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0414-6 · Cited by 473 · see citing works
Continuous evolution of base editors with expanded target compatibility and improved activity
Thuronyi, B. W., Koblan, L. W., Levy, J. M., Yeh, W.-H., Zheng, C., Newby, G. A., Wilson, C., Bhaumik, M., Shubina-Oleinik, O., Holt, J. R., et al. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0193-0 · Cited by 347 · see citing works
α-Ketoglutarate links p53 to cell fate during tumour suppression
Morris, J. P., Yashinskie, J. J., Koche, R., Chandwani, R., Tian, S., Chen, C.-C., Baslan, T., Marinkovic, Z. S., Sánchez-Rivera, F. J., Leach, S. D., et al. (2019) · Nature · doi:10.1038/s41586-019-1577-5 · Cited by 296 · see citing works
Self-citationWebber, B. R., Lonetree, C.-L., Kluesner, M. G., Johnson, M. J., Pomeroy, E. J., Diers, M. D., Lahr, W. S., Draper, G. M., Slipek, N. J., Smeester, B. A., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-13007-6 · Cited by 232 · see citing works
CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank
Geurts, M. H., de Poel, E., Amatngalim, G. D., Oka, R., Meijers, F. M., Kruisselbrink, E., van Mourik, P., Berkers, G., de Winter-de Groot, K. M., Michel, S., et al. (2020) · Cell Stem Cell · doi:10.1016/j.stem.2020.01.019 · Cited by 222 · see citing works
Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens
Morris, J. A., Caragine, C., Daniloski, Z., Domingo, J., Barry, T., Lu, L., Davis, K., Ziosi, M., Glinos, D. A., Hao, S., et al. (2023) · Science · doi:10.1126/science.adh7699 · Cited by 208 · see citing works
Organ-on-a-Chip for Cancer and Immune Organs Modeling
Sun, W., Luo, Z., Lee, J., Kim, H., Lee, K., Tebon, P., Feng, Y., Dokmeci, M. R., Sengupta, S., & Khademhosseini, A. (2019) · Advanced Healthcare Materials · doi:10.1002/adhm.201801363 · Cited by 221 · see citing works
Prediction of the sequence-specific cleavage activity of Cas9 variants
Kim, N., Kim, H. K., Lee, S., Seo, J. H., Choi, J. W., Park, J., Min, S., Yoon, S., Cho, S.-R., & Kim, H. H. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0537-9 · Cited by 198 · see citing works
Adenine base editing in an adult mouse model of tyrosinaemia
Song, C.-Q., Jiang, T., Richter, M., Rhym, L. H., Koblan, L. W., Zafra, M. P., Schatoff, E. M., Doman, J. L., Cao, Y., Dow, L. E., et al. (2019) · Nature Biomedical Engineering · doi:10.1038/s41551-019-0357-8 · Cited by 200 · see citing works
Self-citationEvaluating and enhancing target specificity of gene-editing nucleases and deaminases
Kim, D., Luk, K., Wolfe, S. A., & Kim, J.-S. (2019) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-013118-111730 · Cited by 171 · see citing works
Precision genome editing using cytosine and adenine base editors in mammalian cells
Huang, T. P., Newby, G. A., & Liu, D. R. (2021) · Nature Protocols · doi:10.1038/s41596-020-00450-9 · Cited by 175 · see citing works
An <i>In Vivo Kras</i> Allelic Series Reveals Distinct Phenotypes of Common Oncogenic Variants
Zafra, M. P., Parsons, M. J., Kim, J., Alonso-Curbelo, D., Goswami, S., Schatoff, E. M., Han, T., Katti, A., Fernandez, M. T. C., Wilkinson, J. E., et al. (2020) · Cancer Discovery · doi:10.1158/2159-8290.cd-20-0442 · Cited by 139 · see citing works
Self-citationKim, H. K., Lee, S., Kim, Y., Park, J., Min, S., Choi, J. W., Huang, T. P., Yoon, S., Liu, D. R., & Kim, H. H. (2020) · Nature Biomedical Engineering · doi:10.1038/s41551-019-0505-1 · Cited by 145 · see citing works
Simplified adenine base editors improve adenine base editing efficiency in rice
Hua, K., Tao, X., Liang, W., Zhang, Z., Gou, R., & Zhu, J. (2020) · Plant Biotechnology Journal · doi:10.1111/pbi.13244 · Cited by 108 · see citing works
One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids
Geurts, M. H., Gandhi, S., Boretto, M. G., Akkerman, N., Derks, L. L. M., van Son, G., Celotti, M., Harshuk-Shabso, S., Peci, F., Begthel, H., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-40701-3 · Cited by 66 · see citing works
Yuan, T., Yan, N., Fei, T., Zheng, J., Meng, J., Li, N., Liu, J., Zhang, H., Xie, L., Ying, W., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-25217-y · Cited by 55 · see citing works
Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases
Carlson-Stevermer, J., Das, A., Abdeen, A. A., Fiflis, D., Grindel, B. I., Saxena, S., Akcan, T., Alam, T., Kletzien, H., Kohlenberg, L., et al. (2020) · Nature Communications · doi:10.1038/s41467-020-20065-8 · Cited by 32 · see citing works
Rapid interrogation of cancer cell of origin through CRISPR editing
Feng, W., Cao, Z., Lim, P. X., Zhao, H., Luo, H., Mao, N., Lee, Y. S., Rivera, A. A., Choi, D., Wu, C., et al. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2110344118 · Cited by 25 · see citing works
Self-citationA microglia clonal inflammatory disorder in Alzheimer’s disease
Vicario, R., Fragkogianni, S., Weber, L., Lazarov, T., Hu, Y., Hayashi, S. Y., Craddock, B., Socci, N. D., Alberdi, A., Baako, A., et al. (2025) · eLife · doi:10.7554/elife.96519.3 · Cited by 21 · see citing works
Celotti, M., Derks, L. L. M., van Es, J., van Boxtel, R., Clevers, H., & Geurts, M. H. (2024) · STAR Protocols · doi:10.1016/j.xpro.2024.103189 · Cited by 17 · see citing works
<i>TDP1</i> splice-site mutation causes HAP1 cell hypersensitivity to topoisomerase I inhibition
Goh, C. G., Bader, A. S., Tran, T.-A., Belotserkovskaya, R., D’Alessandro, G., & Jackson, S. P. (2025) · Nucleic Acids Research · doi:10.1093/nar/gkae1163 · Cited by 5 · see citing works
- Authors
- Zafra, M. P., Schatoff, E. M., Katti, A., Foronda, M., Breinig, M., Schweitzer, A. Y., Simon, A., Han, T., Goswami, S., Montgomery, E., Thibado, J., Kastenhuber, E. R., Sánchez-Rivera, F. J., Shi, J., Vakoc, C. R., Lowe, S. W., Tschaharganeh, D. F., & Dow, L. E.
- Affiliations
- Weill Cornell Medicine (US)
- German Cancer Research Center (DE)
- Memorial Sloan Kettering Cancer Center (US)
- Cold Spring Harbor Laboratory (US)
- Corresponding author
- Dow, Lukas E
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2018-07-03
- Volume
- 36
- Issue
- 9
- Pages
- 888-893
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC6130889
- OpenAlex
- W2810023485
- Cited by
- 347
- References
- 8
- Citations by year
- 2026: 26 · 2025: 26 · 2024: 44 · 2023: 49 · 2022: 43 · 2021: 49 · 2020: 47 · 2019: 54 · 2018: 9
- FWCI
- 14.14
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (submittedVersion)
- Institutional Repository of the University of Granada (University of Granada) (submittedVersion, other-oa)
- Funders
- Howard Hughes Medical Institute
- National Institutes of Health (U54OD020355)
- National Cancer Institute (T32CA160001, K22CA181280, R01CA195787, T32CA203702, F31CA192835, F31CA224800, P30CA008748)
- National Institute of General Medical Sciences (T32GM007739)
- 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
- CRISPR base editing, base editors, codon optimization, nuclear localization signal, BE3, single nucleotide variants, intestinal organoids, liver, genome editing efficiency
- MeSH terms
- Gene Editing, Animals, Cell Line, Humans, Genetic Variation, Mice, 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
