CRISPR interference (CRISPRi) for sequence-specific control of gene expression

Journal name:
Nature Protocols
Volume:
8,
Pages:
2180–2196
Year published:
DOI:
doi:10.1038/nprot.2013.132
Published online

Abstract

Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1–2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.

At a glance

Figures

  1. The CRISPRi system for transcription repression in bacteria and human cells.
    Figure 1: The CRISPRi system for transcription repression in bacteria and human cells.

    (a) Depending on the target genomic locus, CRISPRi can block transcription elongation or initiation. When the dCas9-sgRNA complex binds to the nontemplate (NT) DNA strand of the UTR or the protein coding region, it can silence gene expression by blocking the elongating RNAPs. When the dCas9-sgRNA complex binds to the promoter sequence (e.g., the −35 or −10 boxes of the bacterial promoter) or the cis-acting transcription factor binding site (TFBS), it can block transcription initiation by sterically inhibiting the binding of RNAP or transcription factors to the same locus. Silencing of transcription initiation is independent of the targeted DNA strand. (b) The plasmid maps of the sgRNA and dCas9 expression vectors in E. coli. The sgRNA expression plasmid contains a promoter (constitutive—pJ23119 or inducible—pLtetO-1) with an annotated transcription start site (+1), an ampicillin-selectable marker (AmpR) and a ColE1 replication origin. The primer-binding sites for inverse PCR are highlighted. Three restriction sites EcoRI, BglII and BamHI are inserted to flank the sgRNA expression cassette to facilitate BioBrick cloning, so that new sgRNA cassettes can be repeatedly inserted into the striped box region. To ensure efficient transcription termination in E. coli, a strong terminator, rrnB, is added to the 3′ end of the sgRNA expression cassette. The dCas9 plasmid contains an aTc-inducible pLtetO-1 promoter, a strong ribosomal binding site (RBS), a chloramphenicol-resistance marker (CmR) and a p15A replication origin. (c) The plasmid maps used for sgRNA and dCas9 expression in human cells. The sgRNA expression plasmid is based on the pSico lentiviral vector that contains a mouse U6 promoter, an expression cassette consisting of a CMV promoter, a puromycin-resistance gene (Puro) and an mCherry gene for selection or screening of the plasmid, an ampicillin-selectable marker and a ColE1 replication origin for cloning in E. coli cells. Transcription of the U6 promoter starts at the last nucleotide G (red color) within the BstXI restriction site. New sgRNAs can be inserted between the BstXI and XhoI sites. The primer extension and insertion sites for sgRNA cloning are shown. The restriction sites BamHI and NsiI can be used to facilitate the BioBrick cloning: new sgRNA cassettes can be repeatedly inserted into the striped box region using BioBrick. The dCas9 plasmid contains a human codon-optimized dCas9 gene expressed from the murine stem cell virus (MSCV) long terminal repeat (LTR) promoter and is fused to three copies of the SV-40 NLS at the C terminus with a 3-aa linker. The plasmid also contains a puromycin-resistance gene controlled by the PGK promoter.

  2. General workflow for the design, cloning and expression of sgRNAs.
    Figure 2: General workflow for the design, cloning and expression of sgRNAs.

    The orange boxes represent the sgRNA design steps. The green boxes show the cloning steps of sgRNAs for targeting genes in bacteria, and the blue boxes show the cloning of sgRNAs for human cells.

  3. Design of the sgRNAs.
    Figure 3: Design of the sgRNAs.

    (a) The sgRNA is a chimera and consists of three regions: a 20–25-nt-long base-pairing region for specific DNA binding, a 42-nt-long dCas9 handle hairpin for Cas9 protein binding and a 40-nt-long transcription terminator hairpin derived from S. pyogenes. Transcription of sgRNAs should start precisely at its 5′ end. The 12-nt seed region is shaded in orange. (b) The schemes for designing sgRNAs to target the template (T) or nontemplate (NT) DNA strands. When targeting the template DNA strand, the base-pairing region of the sgRNA has the same sequence identity as the transcribed sequence. When targeting the nontemplate DNA strand, the base-pairing region of the sgRNA is the reverse-complement of the transcribed sequence.

  4. Extensions to the base-pairing region with mismatched nucleotides decrease repression activity.
    Figure 4: Extensions to the base-pairing region with mismatched nucleotides decrease repression activity.

    Our results suggest that a precise 5′ end of the sgRNA is required for optimal targeted gene regulation. The sgRNA sequences used for testing the matched and mismatched extensions (additional 5, 10 or 20 nt) are shown on the right side, which are complementary to the mRFP gene. The data are normalized to the repression level achieved when using 20-nt-long sgRNAs (shown as the dotted line). Error bars show s.e.m. from three biological replicates.

  5. Cloning strategy for concatenating multiple sgRNA expression cassettes onto the same plasmid.
    Figure 5: Cloning strategy for concatenating multiple sgRNA expression cassettes onto the same plasmid.

    (a) The cloning method for bacterial sgRNA expression. The donor sgRNA vector is digested using EcoRI and BamHI, and the backbone vector is digested using EcoRI and BglII. Ligation of the two fragments recreates the compatible restriction sites (BglII and BamHI), which can be used for the next round of sgRNA insertion. (b) The cloning method for human sgRNA expression. The main difference from a is that the insert is first PCR amplified to introduce a 5′ BglII site for subsequent BioBrick cloning.

References

  1. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 11731183 (2013).
  2. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 17091712 (2007).
  3. Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331338 (2012).
  4. Wiedenheft, B. et al. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477, 486489 (2011).
  5. Brouns, S.J.J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960964 (2008).
  6. Makarova, K.S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467477 (2011).
  7. Sashital, D.G., Jinek, M. & Doudna, J.A. An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3. Nat. Struct. Mol. Biol. 18, 680687 (2011).
  8. Carte, J., Pfister, N.T., Compton, M.M., Terns, R.M. & Terns, M.P. Binding and cleavage of CRISPR RNA by Cas6. RNA 16, 21812188 (2010).
  9. Karginov, F.V. & Hannon, G.J. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol. Cell 37, 719 (2010).
  10. Sampson, T.R., Saroj, S.D., Llewellyn, A.C., Tzeng, Y.-L. & Weiss, D.S. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 497, 254257 (2013).
  11. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602607 (2011).
  12. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816821 (2012).
  13. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579E2586 (2012).
  14. Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 92759282 (2011).
  15. Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 13901400 (2008).
  16. Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 6771 (2010).
  17. Mojica, F.J.M., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733740 (2009).
  18. Shah, S.A., Erdmann, S., Mojica, F.J.M. & Garrett, R.A. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10, 891899 (2013).
  19. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233239 (2013).
  20. Dicarlo, J.E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 43364343 (2013).
  21. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227229 (2013).
  22. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910918 (2013).
  23. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823826 (2013).
  24. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819823 (2013).
  25. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230232 (2013).
  26. Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013).
  27. Hannon, G.J. RNA interference. Nature 418, 244251 (2002).
  28. Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 2533 (2000).
  29. Segal, D.J. & Barbas, C.F. Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins. Curr. Opin. Biotechnol. 12, 632637 (2001).
  30. Beerli, R.R. & Barbas, C.F. Engineering polydactyl zinc-finger transcription factors. Nat. Biotechnol. 20, 135141 (2002).
  31. Liu, Q., Segal, D.J., Ghiara, J.B. & Barbas, C.F. Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. Proc. Natl. Acad. Sci. USA 94, 55255530 (1997).
  32. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149153 (2011).
  33. Garg, A., Lohmueller, J.J., Silver, P.A. & Armel, T.Z. Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 40, 75847595 (2012).
  34. Sanjana, N.E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171192 (2012).
  35. Kim, D. & Rossi, J. RNAi mechanisms and applications. BioTechniques 44, 613616 (2008).
  36. Klug, A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu. Rev. Biochem. 79, 213231 (2010).
  37. Lopez-Sanchez, M.-J. et al. The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol. Microbiol. 85, 10571071 (2012).
  38. Fischer, S. et al. An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA. J. Biol. Chem. 287, 3335133363 (2012).
  39. 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, 595605 (2012).
  40. Huang, S.H. Inverse polymerase chain reaction. An efficient approach to cloning cDNA ends. Mol. Biotechnol. 2, 1522 (1994).
  41. Quan, J. & Tian, J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat. Protoc. 6, 242251 (2011).
  42. Shetty, R.P., Endy, D. & Knight, T.F. Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2, 5 (2008).
  43. Qi, L., Haurwitz, R.E., Shao, W., Doudna, J.A. & Arkin, A.P. RNA processing enables predictable programming of gene expression. Nat. Biotechnol. 30, 10021006 (2012).
  44. Churchman, L.S. & Weissman, J.S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368373 (2011).
  45. Churchman, L.S. & Weissman, J.S. Native elongating transcript sequencing (NET-seq). Curr. Protoc. Mol. Biol. 4, 14.114.17 (2012).
  46. Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 9961006 (2002).
  47. Meyer, L.R. et al. The UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids Res. 41, D64D69 (2013).
  48. Keseler, I.M. et al. EcoCyc: a comprehensive database of Escherichia coli biology. Nucleic Acids Res. 39, D583D590 (2011).
  49. Bhagwat, M., Young, L. & Robison, R.R. Using BLAT to find sequence similarity in closely related genomes. Curr. Protoc. Bioinformatics 37, 10.8.110.8.24 (2012).
  50. Jiang, H. & Wong, W.H. SeqMap: mapping massive amount of oligonucleotides to the genome. Bioinformatics 24, 23952396 (2008).
  51. Gruber, A.R., Lorenz, R., Bernhart, S.H., Neuböck, R. & Hofacker, I.L. The Vienna RNA websuite. Nucleic Acids Res. 36, W70W74 (2008).
  52. Markham, N.R. & Zuker, M. UNAFold: software for nucleic acid folding and hybridization. Methods Mol. Biol. 453, 331 (2008).
  53. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 34063415 (2003).
  54. Paul, C.P., Good, P.D., Winer, I. & Engelke, D.R. Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505508 (2002).
  55. Lucks, J.B., Qi, L., Mutalik, V.K., Wang, D. & Arkin, A.P. Versatile RNA-sensing transcriptional regulators for engineering genetic networks. Proc. Natl. Acad. Sci. USA 108, 86178622 (2011).
  56. Qi, L., Lucks, J.B., Liu, C.C., Mutalik, V.K. & Arkin, A.P. Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals. Nucleic Acids Res. 40, 57755786 (2012).
  57. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 25, 12031210 (1997).
  58. Ventura, A. et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl. Acad. Sci. USA 101, 1038010385 (2004).
  59. Inoue, H., Nojima, H. & Okayama, H. High efficiency transformation of Escherichia coli with plasmids. Gene 96, 2328 (1990).
  60. Liu, C.C. et al. An adaptor from translational to transcriptional control enables predictable assembly of complex regulation. Nat. Methods 9, 10881094 (2012).
  61. Mutalik, V.K., Qi, L., Guimaraes, J.C., Lucks, J.B. & Arkin, A.P. Rationally designed families of orthogonal RNA regulators of translation. Nat. Chem. Biol. 8, 447454 (2012).
  62. Liu, C.C., Qi, L., Yanofsky, C. & Arkin, A.P. Regulation of transcription by unnatural amino acids. Nat. Biotechnol. 29, 164168 (2011).
  63. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905909 (2005).
  64. Ingolia, N.T., Ghaemmaghami, S., Newman, J.R.S. & Weissman, J.S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218223 (2009).

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Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California, San Francisco (UCSF), San Francisco, California, USA.

    • Matthew H Larson,
    • Luke A Gilbert,
    • Wendell A Lim,
    • Jonathan S Weissman &
    • Lei S Qi
  2. Howard Hughes Medical Institute, UCSF, San Francisco, California, USA.

    • Matthew H Larson,
    • Luke A Gilbert,
    • Wendell A Lim &
    • Jonathan S Weissman
  3. California Institute for Quantitative Biomedical Research, San Francisco, California, USA.

    • Matthew H Larson,
    • Luke A Gilbert,
    • Wendell A Lim,
    • Jonathan S Weissman &
    • Lei S Qi
  4. Bioinformatics Division, Center for Synthetic and Systems Biology, Tsinghua National Laboratory for Information Science and Technology Department of Automation, Tsinghua University, Beijing, China.

    • Xiaowo Wang
  5. UCSF Center for Systems and Synthetic Biology, UCSF, San Francisco, California, USA.

    • Wendell A Lim &
    • Lei S Qi

Contributions

L.S.Q., M.H.L., L.A.G. and X.W. wrote the manuscript. J.S.W., W.A.L. and L.S.Q. supervised the research.

Competing financial interests

The authors have filed a patent related to this work (US provisional patent application number 61/765,576).

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