Abstract
We previously described a protocol for genome engineering of mammalian cultured cells with clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR–Cas9) to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here we are updating this former protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high-efficiency electroporation of optimized CRISPR–Cas9 reagents, screening of single cell-derived clones by automated bright-field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital polymerase chain reaction (PCR) and automated data analysis. Compared with the original protocol, our current procedure (1) substantially increases the efficiency of tag integration, (2) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (3) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with digital PCR. The increased efficiency of the new procedure reduces the number of clones that need to be analyzed in-depth by more than tenfold and yields to more than 26% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 d to 10 d during the overall ~10 week procedure, allowing a single person to process up to five genes in parallel, assuming that validated reagents—for example, PCR primers, digital PCR assays and western blot antibodies—are available.
Key points
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This update to the authors’ previous protocol for genome engineering of mammalian cultured cells with CRISPR–Cas9 to generate homozygous knock-ins of fluorescent tags into endogenous genes improves the method using electroporation, automated imaging and digital PCR screening.
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The updated protocol provides both increased efficiency and throughput.
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Data availability
Raw datasets generated and analyzed during this work are available at https://figshare.com/s/2a35a6156c9a8cf60a86 (https://doi.org/10.6084/m9.figshare.24198609). Any additional data required for research purposes are available from the corresponding authors upon request. Source data are provided with this paper.
Code availability
Computational scripts used for the analysis of dPCR results are available at github (https://github.com/beatrizserrano/CRISPR_updated_protocol/tree/master; refer to https://github.com/beatrizserrano/CRISPR_updated_protocol/blob/master/1_analysisReplicates.Rmd to analyze replicates samples measured with the STILLA device or to https://github.com/beatrizserrano/CRISPR_updated_protocol/blob/master/2_analysis_plates.Rmd to analyze data acquired with the BioRad platform.
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Acknowledgements
We thank N. Daigle for valuable discussions during the entire project and for her great support during the writing of this manuscript. We thank K. Sandvold Beckwith for his important contribution in manuscript revisions. Additionally, we thank our EMBL facilities; the Flow Cytometry Facility, the Advanced Light Microscopy Facility, the Centre for Bioimage Analysis Facility and the Genomics Facility. Furthermore, we highly appreciate the demonstration systems and application specialists from STILLA technologies and Bio-Rad Laboratories.
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Authors and Affiliations
Contributions
M.K. and A.C. planned and executed the experiments. B.S.S. generated the analysis software and performed data analysis. M.K., A.C., B.S.S. and J.E. wrote the manuscript. A.C. and N.R.M. revised the manuscript.
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The authors declare no competing interests.
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Nature Protocols thanks the anonymous reviewer(s) for their contribution to the peer review of this work.
Additional information
Related links
Key references using this protocol
Otsuka, S. et al. Nature 613, 575–581 (2023): https://doi.org/10.1038/s41586-022-05528-w
Koch, B. et al. Nat. Protcoc. 13, 1465–1487 (2018): https://doi.org/10.1038/nprot.2018.042
This protocol is an update to Nat. Protoc. 13, 1465–1487 (2018): https://doi.org/10.1038/nprot.2018.042
Supplementary information
Supplementary Information
Methods, Supplementary Tables 1–10 and Figs. 1–3.
Supplementary Code 1
To be run with dPCR datasets, from BioRad platform.
Supplementary Code 2
To be run with dPCR datasets, from Stilla platform.
Supplementary Code 3
To be run in conjunction with codes 1 and 2.
Source data
Source Data Fig. 3
Fluorescence images in SP8 microscope of representative validated clones.
Source Data Fig. 3
Results from dPCR analysis of CRISPR-edited clones (c and e).
Source Data Fig. 4
Results from dPCR analysis of CRISPR-edited clones (d).
Source Data Fig. 5
Results from dPCR analysis of CRISPR-edited clones (c).
Source Data Box 1
Statistical analysis implemented on the dPCR results for edited clones obtained with the PCRNP method.
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Callegari, A., Kueblbeck, M., Morero, N.R. et al. Rapid generation of homozygous fluorescent knock-in human cells using CRISPR–Cas9 genome editing and validation by automated imaging and digital PCR screening. Nat Protoc 20, 26–66 (2025). https://doi.org/10.1038/s41596-024-01043-6
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DOI: https://doi.org/10.1038/s41596-024-01043-6
