Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a
Cheng Chen,
Yuanxin Liao,
Miao Zhu,
Li Wang,
Xinran Yu,
Meishi Li,
Guangdun Peng
Affiliations
Cheng Chen
Center for Cell Lineage Technology and Engineering, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong-Hong Kong Stem Cell and Regenerative Medicine Research Centre, China-New Zealand Belt and Road Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Yuanxin Liao
Center for Cell Lineage Technology and Engineering, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong-Hong Kong Stem Cell and Regenerative Medicine Research Centre, China-New Zealand Belt and Road Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing, China
Miao Zhu
Center for Cell Lineage Technology and Engineering, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong-Hong Kong Stem Cell and Regenerative Medicine Research Centre, China-New Zealand Belt and Road Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing, China
Li Wang
Center for Cell Lineage Technology and Engineering, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong-Hong Kong Stem Cell and Regenerative Medicine Research Centre, China-New Zealand Belt and Road Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Xinran Yu
Center for Cell Lineage Technology and Engineering, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong-Hong Kong Stem Cell and Regenerative Medicine Research Centre, China-New Zealand Belt and Road Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Meishi Li
Center for Cell Lineage Technology and Engineering, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong-Hong Kong Stem Cell and Regenerative Medicine Research Centre, China-New Zealand Belt and Road Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Guangdun Peng
Center for Cell Lineage Technology and Engineering, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangdong-Hong Kong Joint Laboratory for Stem Cell and Regenerative Medicine, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong-Hong Kong Stem Cell and Regenerative Medicine Research Centre, China-New Zealand Belt and Road Joint Laboratory on Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Corresponding author
Summary: Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.