Base editing in the AUTS2 gene and high-throughput NGS genotyping of clones: a strategy for generating a cellular model

Studying the molecular mechanisms underlying autism spectrum disorders (ASD) requires cellular models capable of capturing cis-regulatory effects and allele-specific gene expression. In this study, we present an approach for generating induced pluripotent stem cells (iPSCs) modified using an adenine base editor (ABE) to introduce synonymous single-nucleotide substitutions in the AUTS2 gene – a candidate involved in ASD pathogenesis. These substitutions serve as allele-specific markers, enabling the tracking of expression differences between normal and rearranged alleles in a cis-regulatory context. We developed a high-efficiency strategy for genotyping clones using amplicon-based next-generation sequencing (NGS). Analysis of over 100 subclones demonstrated that this approach surpasses Sanger sequencing in scalability, sensitivity, and cost-effectiveness. We selected clones with targeted heterozygous substitutions, assessed mosaicism levels, and performed phasing with germline heterozygous variants to confirm monoclonal origin and identify the allele carrying the chromosomal rearrangement. The resulting iPSC lines mark distinct AUTS2 alleles, providing a foundation for analyzing the impact of cis-regulatory elements on gene expression across different cell types. Our findings highlight the practical value of base editors and targeted NGS genotyping in creating cellular models with single-nucleotide substitutions for both basic and applied research.

1. Baux D., Van Goethem C., Ardouin O., Guignard T., Bergougnoux A., Koenig M., Roux A.-F. MobiDetails: online DNA variants interpretation. Eur J Hum Genet. 2021;29(2):356-360. doi 10.1038/s41431-020-00755-z

2. Bennett E.P., Petersen B.L., Johansen I.E., Niu Y., Yang Z., Chamberlain C.A., Met Ö., Wandall H.H., Frödin M. INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels. Nucleic Acids Res. 2020;48(21):11958-11981. doi 10.1093/nar/gkaa975

3. Billon P., Bryant E.E., Joseph S.A., Nambiar T.S., Hayward S.B., Rothstein R., Ciccia A. CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons. Mol Cell. 2017;67(6):1068-1079.e4. doi 10.1016/j.molcel.2017.08.008

4. Chen X., McAndrew M.J., Lapinaite A. Unlocking the secrets of ABEs: the molecular mechanism behind their specificity. Biochem Soc Trans. 2023;51(4):1635-1646. doi 10.1042/BST20221508

5. Davidson C.J., Zeringer E., Champion K.J., Gauthier M.-P., Wang F., Boonyaratanakornkit J., Jones J.R., Schreiber E. Improving the limit of detection for Sanger sequencing: a comparison of methodologies for KRAS variant detection. BioTechniques. 2012;53(3):182-188. doi 10.2144/000113913

6. De Masi C., Spitalieri P., Murdocca M., Novelli G., Sangiuolo F. Application of CRISPR/Cas9 to human-induced pluripotent stem cells: from gene editing to drug discovery. Hum Genomics. 2020; 14(1):25. doi 10.1186/s40246-020-00276-2

7. Gaudelli N.M., Komor A.C., Rees H.A., Packer M.S., Badran A.H., Bryson D.I., Liu D.R. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017;551(7681): 464-471. doi 10.1038/nature24644

8. Geurts M.H., Gandhi S., Boretto M.G., Akkerman N., Derks L.L.M., Van Son G., Celotti M., … Andersson-Rolf A., Chuva De Sousa Lopes S.M., Van Es J.H., Van Boxtel R., Clevers H. One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids. Nat Commun. 2023;14(1):4998. doi 10.1038/s41467-023-40701-3

9. Global Burden of Disease Study 2021 Autism Spectrum Collaborators. The global epidemiology and health burden of the autism spectrum: findings from the Global Burden of Disease Study 2021. Lancet Psychiatry. 2025;12(2):111-121. doi 10.1016/S2215-0366(24)00363-8

10. Gridina M., Lagunov T., Belokopytova P., Torgunakov N., Nuriddinov M., Nurislamov A., Nazarenko L.P., … Filipenko M., Rogaev E., Shilova N.V., Lebedev I.N., Fishman V. Combining chromosome conformation capture and exome sequencing for simultaneous detection of structural and single-nucleotide variants. Genome Med. 2025;17(1):47. doi 10.1186/s13073-025-01471-3

11. Grünewald J., Zhou R., Garcia S.P., Iyer S., Lareau C.A., Aryee M.J., Joung J.K. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature. 2019;569(7756):433- 437. doi 10.1038/s41586-019-1161-z

12. Hwang G.-H., Park J., Lim K., Kim S., Yu J., Yu E., Kim S.-T., Eils R., Kim J.-S., Bae S. Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics. 2018;19(1):542. doi 10.1186/s12859-018-2585-4

13. Jaganathan K., Kyriazopoulou Panagiotopoulou S., McRae J.F., Fazel Darbandi S., Knowles D., Li Y.I., Kosmicki J.A., … Gao H., Kia A., Batzoglou S., Sanders S.J., Farh K.K.-H. Predicting splicing from primary sequence with deep learning. Cell. 2019;176(3):535- 548.e24. doi 10.1016/j.cell.2018.12.015

14. Jin S., Zong Y., Gao Q., Zhu Z., Wang Y., Qin P., Liang C., Wang D., Qiu J.-L., Zhang F., Gao C. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science. 2019; 364(6437):292-295. doi 10.1126/science.aaw7166

15. 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 doublestranded DNA cleavage. Nature. 2016;533(7603):420-424. doi 10.1038/nature17946

16. Liang Y., Chen F., Wang K., Lai L. Base editors: development and applications in biomedicine. Front Med. 2023;17(3):359-387. doi 10.1007/s11684-023-1013-y

17. Lu Z., Huang X. Base editors: a powerful tool for generating animal models of human diseases. Cell Stress. 2018;2(10):242-245. doi 10.15698/cst2018.10.156

18. Rees H.A., Liu D.R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. 2018;19(12): 770-788. doi 10.1038/s41576-018-0059-1

19. Reese M.G., Eeckman F.H., Kulp D., Haussler D. Improved splice site detection in Genie. In: Proceedings of the First Annual International Conference on Computational Molecular Biology (RECOMB ’97). 1997;232-240. doi 10.1145/267521.267766

20. Rowe R.G., Daley G.Q. Induced pluripotent stem cells in disease modelling and drug discovery. Nat Rev Genet. 2019;20(7):377-388. doi 10.1038/s41576-019-0100-z

21. Salnikov P., Belokopytova P., Yan A., Viesná E., Korablev A., Serova I., Lukyanchikova V., Stepanchuk Y., Torgunakov N., Tikhomirov S., Fishman V. Direction and modality of transcription changes caused by TAD boundary disruption in Slc29a3/Unc5b locus depends on tissue-specific epigenetic context. Epigenetics Chromatin. 2025; 18(1):55. doi 10.1186/s13072-025-00618-1

22. Smirnov A.V., Yunusova A.M., Lukyanchikova V.A., Battulin N.R. CRISPR/Cas9, a universal tool for genomic engineering. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J Genet Breed. 2016;20(4): 493-510. doi 10.18699/VJ16.175 (in Russian)

23. Uddin F., Rudin C.M., Sen T. CRISPR gene therapy: applications, limitations, and implications for the future. Front Oncol. 2020;10:1387. doi 10.3389/fonc.2020.01387

24. Yu Y., Leete T.C., Born D.A., Young L., Barrera L.A., Lee S.-J., Rees H.A., Ciaramella G., Gaudelli N.M. Cytosine base editors with minimized unguided DNA and RNA off-target events and high ontarget activity. Nat Commun. 2020;11(1):2052. doi 10.1038/s41467-020-15887-5

25. Zuo E., Sun Y., Wei W., Yuan T., Ying W., Sun H., Yuan L., Steinmetz L.M., Li Y., Yang H. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science. 2019;364(6437):289-292. doi 10.1126/science.aav9973