Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Targeted transcriptional modulation with type I CRISPR–Cas systems in human cells

Abstract

Class 2 CRISPR–Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR–Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR–Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR–Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR–Cas systems for further exploration.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: EcoCascade expression and complex formation in human cells.
Fig. 2: EcoCascade activates transcription of endogenous genes in human cells.
Fig. 3: Genome-wide specificity of EcoCascade–p300 targeted to IL1RN.
Fig. 4: LmoCascade activates transcription of IL1RN gene in human cells.
Fig. 5: LmoCascade represses transcription of HBE1 gene in human cells.

Data availability

The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus56,57 and are accessible through GEO Series Accession number GSE114859. All other relevant raw data are available from the corresponding author upon request.

Code availability

Custom scripts used for ChIP–seq and RNA-seq analysis are available upon request.

References

  1. 1.

    Gaj, T., Gersbach, C. A. & Barbas, C. F. 3rd ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Barrangou, R. & Doudna, J. A. Applications of CRISPR technologies in research and beyond. Nat. Biotechnol. 34, 933–941 (2016).

    CAS  PubMed  Google Scholar 

  3. 3.

    Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Majumdar, S. et al. Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus. RNA 21, 1147–1158 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).

    CAS  PubMed  Google Scholar 

  9. 9.

    Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

    CAS  PubMed  Google Scholar 

  10. 10.

    Mulepati, S. & Bailey, S. In vitro reconstitution of an Escherichia coli RNA-guided immune system reveals unidirectional, ATP-dependent degradation of DNA target. J. Biol. Chem. 288, 22184–22192 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Jore, M. M. et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat. Struct. Mol. Biol. 18, 529–536 (2011).

    CAS  PubMed  Google Scholar 

  13. 13.

    Wiedenheft, B. et al. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477, 486–489 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Jackson, R. N. et al. Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science 345, 1473–1479 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Zhao, H. et al. Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli. Nature 515, 147–150 (2014).

    CAS  PubMed  Google Scholar 

  16. 16.

    Hayes, R. P. et al. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli. Nature 530, 499–503 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Gesner, E. M., Schellenberg, M. J., Garside, E. L., George, M. M. & Macmillan, A. M. Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat. Struct. Mol. Biol. 18, 688–692 (2011).

    CAS  PubMed  Google Scholar 

  18. 18.

    Beloglazova, N. et al. CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli. Nucleic Acids Res. 43, 530–543 (2015).

    CAS  PubMed  Google Scholar 

  19. 19.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Luo, M. L., Mullis, A. S., Leenay, R. T. & Beisel, C. L. Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res. 43, 674–681 (2015).

    CAS  PubMed  Google Scholar 

  26. 26.

    Westra, E. R. et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell 46, 595–605 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Hochstrasser, M. L., Taylor, D. W., Kornfeld, J. E., Nogales, E. & Doudna, J. A. DNA targeting by a minimal CRISPR RNA-guided Cascade. Mol. Cell 63, 840–851 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Leenay, R. T. et al. Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol. Cell 62, 137–147 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol 32, 670–676 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol 32, 677–683 (2014).

    CAS  PubMed  Google Scholar 

  31. 31.

    Thakore, P. I. et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Bolukbasi, M. F., Gupta, A. & Wolfe, S. A. Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat. Methods 13, 41–50 (2016).

    CAS  PubMed  Google Scholar 

  33. 33.

    Di, H. et al. Comparative analysis of CRISPR loci in different Listeria monocytogenes lineages. Biochem. Biophys. Res. Commun. 454, 399–403 (2014).

    CAS  PubMed  Google Scholar 

  34. 34.

    Rath, D., Amlinger, L., Hoekzema, M., Devulapally, P. R. & Lundgren, M. Efficient programmable gene silencing by Cascade. Nucleic Acids Res. 43, 237–246 (2015).

    CAS  PubMed  Google Scholar 

  35. 35.

    Margolin, J. F. et al. Kruppel-associated boxes are potent transcriptional repression domains. Proc. Natl Acad. Sci. USA 91, 4509–4513 (1994).

    CAS  PubMed  Google Scholar 

  36. 36.

    Klann, T. S. et al. CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Sinkunas, T. et al. In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. EMBO J. 32, 385–394 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Chen, F. et al. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting. Nat. Commun. 8, 14958 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e214 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Gleditzsch, D. et al. Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system. Nucleic Acids Res. 44, 5872–5882 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Kleinstiver, B. P. et al. Monomeric site-specific nucleases for genome editing. Proc. Natl Acad. Sci. USA 109, 8061–8066 (2012).

    CAS  PubMed  Google Scholar 

  42. 42.

    Beurdeley, M. et al. Compact designer TALENs for efficient genome engineering. Nat. Commun. 4, 1762 (2013).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Kleinstiver, B. P. et al. The I-TevI nuclease and linker domains contribute to the specificity of monomeric TALENs. G3 (Bethesda) 4, 1155–1165 (2014).

    CAS  Google Scholar 

  44. 44.

    Dolan, A. E. et al. Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using Type I CRISPR-Cas. Mol Cell 74, 936–950 (2019).

    CAS  PubMed  Google Scholar 

  45. 45.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016).

    PubMed  Google Scholar 

  48. 48.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009).

    PubMed  PubMed Central  Google Scholar 

  51. 51.

    Consortium, E. P. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

    Google Scholar 

  52. 52.

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).

    CAS  PubMed  Google Scholar 

  55. 55.

    Pinello, L. et al. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat. Biotechnol. 34, 695–697 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Edgar, R., Domrachev, M. & Lash, A. E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Barrett, T. et al. NCBI GEO: archive for functional genomics data sets—update. Nucleic Acids Res. 41, D991–D995 (2013).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Oliver, I. Hilton and D. D. Kocak for technical assistance and helpful comments and D. Ousterout for valuable discussions. This work was supported by Locus Biosciences, an Allen Distinguished Investigator Award from the Paul G. Allen Frontiers Group, the Thorek Memorial Foundation, US National Institutes of Health (NIH) grant R01DA036865, an NIH Director’s New Innovator Award (DP2OD008586), a US National Science Foundation (NSF) Faculty Early Career Development (CAREER) Award (CBET-1151035), NSF grant DMR-1709527, and an NSF Emerging Frontiers in Research and Innovation (EFRI) Award (EFMA-1830957). A.P.-O. was supported by a Pfizer-NCBiotech Distinguished Postdoctoral Fellowship. J.B.B. was supported by an NIH Biotechnology Training Grant (T32GM008555) and Predoctoral Fellowship (F31NS105419). R.B. was supported by internal funds from NC State University. C.L.B. was supported by an NIH Maximizing Investigator’s Research Award (1R35GM119561-01).

Author information

Affiliations

Authors

Contributions

A.P.-O., J.B.B and C.A.G. designed the experiments. A.P.-O., J.B.B., M.M.L., K.J.M., T.S.K., K.A.G., M.J.S. and L.C.B performed the experiments. A.P.-O., J.B.B., C.E.N., A.B., T.E.R. and C.A.G. analyzed the data. C.L.B. and R.B. provided Cascade sequences. A.P.-O. and C.A.G. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Charles A. Gersbach.

Ethics declarations

Competing interests

C.A.G., A.P.-O., R.B. and C.L.B. have filed patent applications related to genome engineering with type I CRISPR systems. T.S.K. is a co-founder of, and advisor to, Element Genomics. C.A.G. is a co-founder of, and advisor to, Locus Biosciences and Element Genomics, and an advisor to Sarepta Therapeutics. R.B. is a co-founder and Scientific Advisory Board member of Locus Biosciences and Intellia Therapeutics. C.L.B. is a co-founder and Scientific Advisory Board member of Locus Biosciences.

Additional information

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

Integrated supplementary information

Supplementary Figure 1 Activation is dependent on targeting EcoCascade-p300 to genomic loci.

(a) Schematic of the IL1RN locus along with IL1RN crRNA target sites. (b) Relative IL1RN expression following co-transfection of individual crRNAs and EcoCascade with Cas6-p300, unless otherwise noted. All samples processed at 3 days post-transfection. (Tukey-test following logarithmic transformation, **P<0.001, n.s. P>0.05 compared to Ctrl crRNA, n=3 biological independent samples; error bars, SEM). Numbers above bars indicate mean relative expression. TSS, transcription start site; Ctrl crRNA, Control crRNA; n.s., non-significant.

Supplementary Figure 2 Heterologous transfection of Cas constructs reveals optimized transactivation activity.

(a) Schematic of the IL1RN locus along with IL1RN crRNA target sites. (b) Relative IL1RN expression following co-transfection of 100 ng individual crRNAs and 500 ng total EcoCascade with Cas6-p300 in various ratios. All samples processed at 3 days post transfection. (Tukey-test, *P<0.05, n=3 biological independent samples; error bars, SEM). Bars indicate mean relative expression. TSS, Transcription start site.

Supplementary Figure 3 Programmable EcoCascade-p300 interacts with endogenous DNA target.

(a) Schematic of the IL1RN locus along with IL1RN crRNA and sgRNA target sites. Optimal dCas9 sgRNA-3 was identified in previous work (Nat Biotechnol, 510-517, 2015). IL1RN amplicons are shown in corresponding locations. (b) ChIP-qPCR enrichment following co-transfection of individual crRNAs with EcoCascade and Cas6-p300. IP performed with α-Flag and qPCR performed with primers for amplicon regions designated in a. All samples processed at 3 days post-transfection. (Tukey-test among conditions for each ChIP-qPCR region, *P<0.05 compared to Ctrl crRNA, †P<0.05 compared to dCas9-p300+g3, n=3 biological independent samples; error bars, SEM). Bars indicate mean relative expression. TSS, Transcription start site; Ctrl crRNA, Control crRNA; ChIP, chromatin immunoprecipitation; qPCR, g3, sgRNA-3.

Supplementary Figure 4 Targeted EcoCascade-mediated transactivation can be achieved by tethering different activation domains to Cas6.

(a) Schematic of the IL1RN locus along with IL1RN crRNA and sgRNA target sites. (b) Relative IL1RN expression following co-transfection of individual crRNAs and EcoCascade with Cas6-p300 or Cas6-VPR fusions. (c) Relative IL1RN expression following co-transfection of individual sgRNAs with dCas9-p300 or dCas9-VPR fusions. All samples processed at 3 days post transfection. (Tukey-test, **P<0.001 compared to Ctrl crRNA, ††P<0.001 compared to same crRNA, n=3 biological independent samples; error bars, SEM). Bars indicate mean relative expression. TSS, transcription start site; Ctrl crRNA, Control crRNA; VPR, VP64-p65-Rta tripartite activator.

Supplementary Figure 5 Targeted multiplexed transactivation by EcoCascade-p300 at the human IL1RN and HBG loci.

(a) Schematic of the IL1RN and HBG loci along with IL1RN and HBG crRNA target sites. (b) Schematic representation of CRISPR array containing multiple crRNA spacers that target both IL1RN and HBG. Cleavage of the crRNA transcript at the sites indicated with blue arrows yields mature crRNAs. (c) Relative IL1RN and HBG expression following co-transfection of individual crRNAs or crRNA arrays and EcoCascade with Cas6-p300. All samples processed at 3 days post-transfection. (Tukey-test, **P<0.001 compared to Ctrl crRNA, n=3 biological independent samples; error bars, SEM). Bars indicate mean relative expression. TSS, transcription start site; Ctrl crRNA, Control crRNA.

Supplementary Figure 6 Genome-wide specificity of EcoCascade-p300 targeted to IL1RN.

(a) MA plot for ChIP-seq differential binding of Flag-tagged EcoCascade-p300 targeted to the IL1RN promoter with cr26 compared with binding of EcoCascade-p300 targeted to the IL1RN promoter with cr25 in HEK293T cells. Red data points indicate FDR < 0.001 by differential DESeq2 analysis using Wald test p-values. (b) MA plot for the differential expression of EcoCascade-p300 targeted by cr26 versus EcoCascade targeted by cr26 in HEK293T cells. Red data points indicate FDR < 0.01 by differential expression analysis using Wald test p-values. (n=3 biological independent samples).

Supplementary Figure 7 Transactivation of LmoCascade-p300 targeted to HBG in human cells.

(a) Schematic of the HBG loci along with HBG crRNA target sites. (b) Relative HBG expression following co-transfection of individual crRNAs or crRNA arrays and LmoCascade with Cas6-p300. All samples processed at 3 days post-transfection. (Tukey-test, **P<0.001 compared to Ctrl crRNA, *P<0.05 compared to Ctrl crRNA n=3 biological independent samples; error bars, SEM). Numbers above bars indicate mean relative expression. TSS, transcription start site; Ctrl crRNA, Control crRNA.

Supplementary Figure 8 Stable LmoCascade-KRAB expression achieved by co-transduction and selection in K562-HBE1-mCherry reporter cell line.

(a) Schematic of lentiviral expression constructs. (b) Western blot showing co-expression of LmoCascade following co-transduction of lentivirus encoding the Flag-tagged LmoCascade subunits and Cas6-KRAB-2A-Blast in K562-HBE1-mCherry cells, selection with blasticidin S, and clonal expansion. Clone 2 was expanded for additional experiments.

Supplementary Figure 9 Targeted EcoCascade-mediated transactivation can be enhanced by co-targeting with dCas9.

(a) Schematic of the IL1RN locus along with IL1RN crRNA and sgRNA target sites. (b) Relative IL1RN expression following co-transfection of individual crRNAs, EcoCascade with Cas6-p300, dCas9 (no effector), and individual sgRNAs, where indicated. All samples processed at 3 days post transfection. (Tukey-test following logarithmic transformation, **P<0.001 compared to EcoCascade-p300 with Ctrl crRNA, ††P<0.001 compared to EcoCascade-p300 with cr19, n=3 biological independent samples; error bars, SEM). Bars indicate mean relative expression. TSS, transcription start site; Ctrl crRNA, Control crRNA; g2, single-guide RNA-2; g3, single-guide RNA-3.

Supplementary Figure 10 Targeted LmoCascade editing can be achieved by tethering I-TevI monomeric endonuclease domains to Cas6.

(a) Schematic of the IL1RN locus along with IL1RN crRNA target sites. (b) I-TevI-Cas6 effectors of different lengths driven by human cytomegalovirus (CMV) promoter. (c) Quantification of percent genomic DNA editing modifications generated following co-transfection of individual crRNAs and LmoCascade with I-TevI(184)-Cas6 or I-TevI(206)-Cas6 fusions in HEK293T cells. (d) Schematic of genomic DNA modifications at IL1RN cr14 target site and representative sequences of mutated alleles identified from deep sequencing analysis of HEK293T bulk cell population co-transfected with LmoCascade and I-TevI(184)-Cas6. I-TevI 5’-CNNNG-3’ nuclease motifs highlighted in orange. All samples processed at 4 days post transfection. (n=1 biological independent sample). TSS, transcription start site; NT, No treatment.

Supplementary Figure 11 Illustration of EcoCascade crRNA cloning scheme for expression in mammalian cells.

(a) Schematic representation of E. coli K-12 processed crRNA with 5’ PAM recognition and base pairing at the DNA target loci. (b) Engineering of pre-processed crRNA driven by U6 promoter for expression and processing in mammalian cells. (c) Illustration of pAPcrRNA_Eco cloning plasmid digested with SacII and XhoI. To insert repeat-spacer pairs, forward and reverse oligonucleotides encoding the palindromic repeat and crRNA spacers were synthesized. For each crRNA, the 3’ XhoI site results in mismatched nucleotides between the spacer and target sequences. Annealed oligonucleotide pairs can be annealed, 5’ phosphorylated with PNK and ligated into digested pAPcrRNA_Eco. Oligo, oligonucleotide.

Supplementary Figure 12 Illustration of LmoCascade crRNA cloning scheme for expression in mammalian cells.

(a) Schematic representation of L. monocytogenes Finland_1998 processed crRNA with 5’ PAM recognition and base pairing at the DNA target loci. (b) Engineering of pre-processed crRNA driven by U6 promoter for expression and processing in mammalian cells. (c) Illustration of pAPcrRNA_Lmo cloning plasmid digested with SacII and AgeI. To insert repeat-spacer pairs, forward and reverse oligonucleotides encoding the palindromic repeat and crRNA spacers were synthesized. For each crRNA, the 3’ AgeI site results in mismatched nucleotides between the spacer and target sequences. Annealed oligonucleotide pairs can be annealed, 5’ phosphorylated with PNK and ligated into digested pAPcrRNA_Lmo. Oligo, oligonucleotide.

Supplementary information

Supplementary Information

Supplementary Figures 1-12, Supplementary Tables 1-5, and Supplementary Note

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pickar-Oliver, A., Black, J.B., Lewis, M.M. et al. Targeted transcriptional modulation with type I CRISPR–Cas systems in human cells. Nat Biotechnol 37, 1493–1501 (2019). https://doi.org/10.1038/s41587-019-0235-7

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing