Supplementary Table S2 contains a list of the plasmids used in this study. Plasmid pDB98 (23 (link)) was used as the source for crRNA guides in S. pneumoniae DB17. This plasmid is a derivative of pLZ12spec (33 (link)) that carries a minimal CRISPR array from S. pyogenes SF370 CRISPR02 containing the leader sequence and two repeats separated by a spacer carrying two BsaI restriction sites for the easy cloning of new spacers. Spacers were cloned by digestion with BsaI, and ligation of annealed oligonucleotides designed as follow: 5′-aaac+(target sequence)+g-3′ and 5′-aaaac+(reverse complement of the target sequence)-3′, where the target sequence is 30 nt and is followed by a functional PAM (NGG). A list of all spacers tested in this study is provided in Supplementary Table S3.
Plasmid pCas9 was generated in a previous study (23 (link)). Plasmid pdCas9 was constructed by introducing D10A and H840A mutations to cas9 on pCas9 through amplification of this plasmid with primers B337/B340 and B338/B339, followed by Gibson assembly (34 (link)) of the two products. Both plasmids contain a minimal CRISPR array with BsaI sites for the cloning of new spacers using complementary oligonucleotides. The in-frame deletion of dcas9 on pdCas9 was achieved by amplification of the plasmid with primers B544/B545 and Gibson assembly of the resulting products.
Fusion of the ω subunit (rpoZ) to dCas9 was achieved by amplification of pdCas9 with primers B441/W551 or B446/W552, and amplification of rpoZ with primers B442/W550 or W553/B448 to create the C- or N-terminal ω fusions, respectively, followed by Gibson assembly. Plasmid pWJ66 carries the C-terminal fusion and pWJ68 the N-terminal fusion. To measure induction in E. coli KS1ΔZ, a chloramphenicol-resistant strain, the plasmid resistance was changed to spectinomycin. Plasmids pWJ66 and pWJ68 were amplified with oligos H001/H002, and the spectinomycin resistance gene was amplified from pSWKspec with oligos H003/H004; Gibson assembly of the PCR products generated plasmids pDB191 and pDB192.
Plasmid pDB127 was constructed by amplification of gfp-mut2 (35 (link)) with primers B368/B371, followed by digestion with EcoRI and BamHI, and ligation together with the annealed oligonucleotides B369/B370 in the pZS24-MCS1 (36 (link)) vector cut with XhoI and BamHI. The sequence of the PAM-rich promoter carried by pDB127 is provided in the Supplementary Sequences.
Plasmids pWJ89, pWJ96 and pWJ97 were constructed by changing the promoter of gfp-mut2 on pDB127 for biobrick promoters BBa_J23117, BBa_J23116 and BBa_J23110, respectively (http://partsregistry.org). The region upstream of the promoter was changed to include multiple NGG PAM sequences on both strands. Full sequences of these promoters are provided in the Supplementary Sequences.