a, Mechanism of CRISPRi repression. A sgRNA–dCas9 complex binds to DNA by base pairing and sterically blocks the progression of RNA polymerase (RNAP), reducing gene expression. b, Mobile-CRISPRi modularity. Individual modules are flanked by unique restriction sites, which can be used for cloning sgRNA libraries or exchanging other components (for example, an antibiotic resistance marker (ABR) or the dcas9 promoter). c, Strain construction using Mobile-CRISPRi. Top: a Tn7 transposon carrying CRISPRi components (shown in b) and a plasmid containing Tn7 transposition genes are transferred to recipient bacteria by tri-parental mating. Donor cells contain a chromosomal copy of the RP4 transfer machinery used to mobilize the Tn7 plasmids. Once inside the recipient cell, Tn7 transposition proteins integrate the CRISPRi DNA (purple) flanked by left and right Tn7 end sequences (green) into the recipient genome downstream of the glmS gene. Selection on antibiotic plates lacking DAP eliminates the E. coli donors and retains recipients with an integrated CRISPRi system. Bottom: an ICE element carrying CRISPRi components is transferred to recipient bacteria by bi-parental mating. Once inside the recipient cell, the ICE integrase inserts ICE into trnS-leu2. Double antibiotic plates that select for ICE and for the intrinsic resistance of the recipient strain (streptomycin resistance in this work) are used to identify recipients with an integrated CRISPRi system.