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

Journal name:
Nature Protocols
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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


  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.


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Author information


  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


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.

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

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