A multi-kingdom genetic barcoding system for precise clone isolation

Soh Ishiguro^#¹, Kana Ishida^#², Rina C Sakata^#¹, Minori Ichiraku³, Ren Takimoto¹, Rina Yogo¹, Yusuke Kijima¹, Hideto Mori⁴, Mamoru Tanaka⁵, Samuel King¹, Shoko Tarumoto⁶, Taro Tsujimura⁶, Omar Bashth¹, Nanami Masuyama^{1

7

8}, Arman Adel¹, Hiromi Toyoshima⁵, Motoaki Seki⁵, Ju Hee Oh⁹, Anne-Sophie Archambault⁹, Keiji Nishida^{10

11}, Akihiko Kondo^{9

10

11

12}, Satoru Kuhara¹³, Hiroyuki Aburatani⁵, Ramon I Klein Geltink⁹, Takuya Yamamoto^{3

6}, Nika Shakiba^{1

4}, Yasuhiro Takashima³, Nozomu Yachie^{14

15

16}

Affiliations

¹ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada.
² Spiber Inc, Tsuruoka, Japan.
³ Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
⁴ Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), The University of Osaka, Osaka, Japan.
⁵ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
⁶ Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan.
⁷ Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
⁸ Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Japan.
⁹ BC Children's Hospital Research Institute, Department of Pathology and Laboratory Medicine, The University of British Columbia, Vancouver, British Columbia, Canada.
¹⁰ Engineering Biology Research Center, Kobe University, Kobe, Japan.
¹¹ Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
¹² Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
¹³ Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
¹⁴ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada. nozomu.yachie@ubc.ca.
¹⁵ Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), The University of Osaka, Osaka, Japan. nozomu.yachie@ubc.ca.
¹⁶ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. nozomu.yachie@ubc.ca.

^# Contributed equally.

PMID: 40399693
DOI: 10.1038/s41587-025-02649-1

A multi-kingdom genetic barcoding system for precise clone isolation

Soh Ishiguro et al. Nat Biotechnol. 2025.

. 2025 May 21.

doi: 10.1038/s41587-025-02649-1. Online ahead of print.

Authors

Affiliations

¹ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada.
² Spiber Inc, Tsuruoka, Japan.
³ Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
⁴ Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), The University of Osaka, Osaka, Japan.
⁵ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
⁶ Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan.
⁷ Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
⁸ Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Japan.
⁹ BC Children's Hospital Research Institute, Department of Pathology and Laboratory Medicine, The University of British Columbia, Vancouver, British Columbia, Canada.
¹⁰ Engineering Biology Research Center, Kobe University, Kobe, Japan.
¹¹ Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
¹² Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
¹³ Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
¹⁴ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada. nozomu.yachie@ubc.ca.
¹⁵ Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), The University of Osaka, Osaka, Japan. nozomu.yachie@ubc.ca.
¹⁶ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. nozomu.yachie@ubc.ca.

^# Contributed equally.

PMID: 40399693
DOI: 10.1038/s41587-025-02649-1

Abstract

Cell-tagging strategies with DNA barcodes have enabled the analysis of clone size dynamics and clone-restricted transcriptomic landscapes in heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here we present a multi-kingdom genetic barcoding system, CloneSelect, which enables a target cell clone to be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first stably tagged with DNA barcodes and propagated so that their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored during the experiment using CRISPR base editing. CloneSelect is scalable and compatible with single-cell RNA sequencing. We demonstrate the versatility of CloneSelect in human embryonic kidney 293T cells, mouse embryonic stem cells, human pluripotent stem cells, yeast cells and bacterial cells.

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Conflict of interest statement

Competing interests: K.I. is an employee of Spiber Inc. The other authors declare no competing interests.

References

1. Bhang, H. E. et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nat. Med. 21, 440–448 (2015). - PubMed - DOI
1. Hata, A. N. et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat. Med. 22, 262–269 (2016). - PubMed - PMC - DOI
1. Ben-David, U. et al. Genetic and transcriptional evolution alters cancer cell line drug response. Nature 560, 325–330 (2018). - PubMed - PMC - DOI
1. Nguyen, L. V. et al. Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells. Nature 528, 267–271 (2015). - PubMed - DOI
1. Venkataram, S. et al. Development of a comprehensive genotype-to-fitness map of adaptation-driving mutations in yeast. Cell 166, 1585–1596.e22 (2016). - PubMed - PMC - DOI

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A multi-kingdom genetic barcoding system for precise clone isolation

Affiliations

A multi-kingdom genetic barcoding system for precise clone isolation

Authors

Affiliations

Abstract

Conflict of interest statement

References

LinkOut - more resources

Full Text Sources

Research Materials