CRISPRI-seq_Streptococcuspneumoniae_competence_1: + 1 mM IPTG + CSP c
The pooled CRISPRi library containing 1499 sgRNAs targeting all the transcription start sites in S. pneumoniae D39V strain (PMID: 33120116) was used to identify essential genes during competence. A pre-culture of the pooled library was grown at in C+Y medium pH 6.8 to avoid natural competence induction, at 37 C until OD595 nm of 0.1 (Figure 1A). Then, the pre-culture was diluted at OD595 nm 0.005 in presence or absence of 1 mM IPTG to induce the expression of dCas9. After 2 h (OD595 nm ~ 0.1) of library induction, 100 ng/ml CSP1 was added in half of the samples to induce competence. Cells were incubated at 37 C until OD595 nm 0.4, and DNA was isolated using a commercial kit (Promega). Preparation of the Ilumina library by one-step PCR with commercial oligos and library sequencing were performed following the manufacturer instructions and described previously (PMID: 34997243).