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An efficient KRAB domain for CRISPRi applications in human cells

Abstract

Clustered regularly interspaced short palindromic repeat interference (CRISPRi), based on the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor, is a powerful platform for silencing gene expression. However, it suffers from incomplete silencing of target genes. We assayed 57 KRAB domains for their repressive potency and identified the ZIM3 KRAB domain as an exceptionally potent repressor. We establish that ZIM3 KRAB–dCas9 fusion silences gene expression more efficiently than existing platforms.

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Fig. 1: Identification of a potent KRAB-domain repressor.
Fig. 2: ZIM3 KRAB–dCas9 outperforms current CRISPRi platforms.

Data availability

All raw data generated during this project are available from the authors upon request. RNA sequencing data are available at NCBI SRA under BioProject no. PRJNA640683. ZIM3 KRAB lentiviral plasmids are available at Addgene with IDs 154472 (pLX303-ZIM3-KRAB-dCas9), 154473 (pHR-UCOE-SFFV-dCas9-mCherry-ZIM3-KRAB), 154474 (pLX303-ABI1-dCas9) and 154761 (pLX301-ZIM3-KRAB-PYL1). All other plasmids are available from the authors upon request. Source data are provided with this paper.

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Acknowledgements

We thank C. Mogg, F. Nowlan and A. Li for their expert help during the project and T. Hughes and F. Schmitges for complementary DNA clones encoding KRAB-domain proteins. We also acknowledge the Donnelly Sequencing Centre for their help with RNA-seq and the Lunenfeld-Tanenbaum Research Institute Sequencing Facility for gRNA library sequencing. This project was supported by Natural Sciences and Engineering Research Council (NSERC) graduate fellowship to D.S., University of Toronto’s Medicine by Design initiative postdoctoral fellowship to H.L. University of Toronto Open Fellowship Award to N.A. and Ontario Early Researcher Award, Tier 2 Canada Research Chair and CIFAR Azrieli Global Scholar Award to M.T. The research in M.T.’s laboratory is supported by Canadian Institutes of Health Research (CIHR), NSERC, CIFAR, Canada Research Chairs program and the University of Toronto’s Medicine by Design initiative.

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Affiliations

Authors

Contributions

N.A. and M.T. designed all experiments. D.S. performed all LUMIER assays and H.L. carried out all experiments with DTA and generated the HBEGF knockout cells. N.A. performed all other experiments. All authors analyzed the data. The manuscript was written by N.A. and M.T., with input from all coauthors.

Corresponding author

Correspondence to Mikko Taipale.

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The authors have submitted an intellectual property claim for using the technology for therapeutic applications.

Additional information

Peer review information Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Characterization of a potent KRAB domain repressor.

a, Comparison of KRAB-dCas9 fusion potency and expression levels. Expression was measured by Western blotting 21 days after infection. KOX1v2, which has also been used in previous studies30, contains a V38L point mutation in the KRAB domain but is otherwise identical to the canonical KOX1. EGFP fluorescence was normalized to that of reporter cells expressing Nanoluc-dCas9 targeted to the promoter. n = three independent lentiviral infections b, Correlation between normalized EGFP fluorescence and DsRed fluorescence in K562 reporter cells stably infected with KRAB-dCas9-P2A-DsRed constructs. EGFP fluorescence was normalized to reporter cells expressing gRNA only. c, Correlation between the efficacy of KRAB-dCas9 fusions in the HEK293T reporter cell line and the K562 reporter cell line. Correlation was calculated using log10-transformed values. d, Flow cytometry histograms showing the distribution of EGFP fluorescence in HEK293T reporter cells expressing the indicated constructs. e, Comparison of different KOX1 KRAB and ZIM3 KRAB domain constructs. Indicated KRAB-dCas9 fusions were recruited to the CD81 promoter (left) in A375 and HEK293T cells or to the SV40-EGFP reporter in HEK293T cells (right) and EGFP fluorescence was measured by flow cytometry. KOX1 (1–75) pLX311 is a lentiviral construct used in previous CRISPRi studies6. The numbers in construct labels refer to the amino acids of KOX1 (Uniprot P21506-1) and ZIM3 (Q96PE6-1) included in the fusion.

Source data

Extended Data Fig. 2 Functional analysis of KRAB domains.

a, Correlation between the repressive activity of KRAB domains and the number of TRIM28 peptides recovered in by AP-MS for full-length KRAB zinc finger proteins. Top, comparison to BioPlex 3.0 dataset15. Bottom, comparison to Helleboid et al. dataset14. Correlations were calculated from log10-transformed data. b, Top, correlation between the repressive activity of KRAB domains and their interaction with HP1α as measured by LUMIER assay in HEK293T cells. Interaction strength is shown as fold change over negative control bait (EGFP). Bottom, correlation between bait expression level (as measured by ELISA) and TRIM28 or HP1α interaction. Expression was normalized to that of empty (non-transfected) wells and interaction strength is shown as fold change over negative control bait (EGFP). All values shown are an average of two biological replicates. Correlations were calculated from log10-transformed data. c, Expression level of PYL1 fusion constructs in stable HEK293T SV40-EGFP reporter cells was assessed with qRT-PCR. The expression level was normalized to that of Firefly luciferase (Fluc) fused to PYL1. d, EGFP silencing was induced by treating KRAB-PYL1 and ABI1-dCas9 expressing SV40-EGFP reporter cells with 100 µM ABA. After 40 days of ABA treatment, ABA was washed off and EGFP expression followed with flow cytometry for another 48 days. EGFP fluorescence was normalized to that of Firefly luciferase-dCas9 fusion similarly recruited to the reporter. The values shown are from a single biological replicate.

Source data

Extended Data Fig. 3 Characterization of ZIM3 KRAB-dCas9 CRISPRi platform.

a, dCas9 fusions were recruited to BLM, ARPC2, or MET promoters with one or two gRNAs for seven days and mRNA expression quantified with qRT-PCR. The expression level of the target was normalized to that of cells expressing Nanoluc-dCas9 targeted to the promoter. Two-tailed Student’s t-test with Bonferroni correction for multiple hypotheses was used to assess statistical significance. n = three or two independent lentiviral infections, as indicated b, Expression level of dCas9 fusion proteins was assayed by western blotting with a Cas9-specific antibody. c, RNA-seq analysis of HEK293T SV40-EGFP reporter cell line expressing indicated dCas9 fusions targeted to the SV40 promoter. Differentially expressed transcripts (absolute log2 fold-change > 0.5 and FDR < 0.05) are shown as solid circles. d, Receiver operating characteristics curves for CRISPRi dropout screens reported here and in Yeo et al. 5. Only genes that were targeted by Yeo et al. are included in the dataset. e, Number of essential genes identified in dropout CRISPRi screens with indicated dCas9 fusions. f, TRIM28 protein expression level in HEK293T cells, K562 cells and A375 cells was assayed by Western blotting.

Source data

Extended Data Fig. 4 Example of the gating strategy for flow cytometry experiments.

Samples were first gated for live cells and single cells, then for EBFP expression (expressed by the gRNA plasmid), and finally for DsRed expression (expressed by the KRAB-dCas9-P2A-DsRed construct). Median EGFP fluorescence was calculated from the final gated population.

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Tables 1–4

Source data

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Alerasool, N., Segal, D., Lee, H. et al. An efficient KRAB domain for CRISPRi applications in human cells. Nat Methods 17, 1093–1096 (2020). https://doi.org/10.1038/s41592-020-0966-x

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