Background

Various CRISPR interference systems have been developed for use in organisms from bacteria to eukaryotes. For those who are considering to use CRISPRi for bacteria, we provide the following background information on our tCRISPRi system (Li et al., 2016) and its comparison with other CRISPRi systems.

Morgan-Kiss et al. (2002) developed the plasmid-based, dose-inducible promoter pBAD. Their system allows tunable expression of a protein from the pBAD promoter, dependent upon arabinose levels. The arabinose transporter genes araE and araFGH are inactive in the strain. Their strain also has two copies of lacY; the wild-type lacY on the chromosome and a mutant lactose transporter lacY A177C on a plasmid. The LacY A177C function allows arabinose to diffuse into the cell, and thus, the pBAD induction level is precisely controlled by the concentration of the supplied arabinose in the medium (Morgan-Kiss, 2002).

Our tCRISPRi strain contains only the mutant gene lacY A177C (Morgan-Kiss, 2002), which is expressed from the lac operon constitutively because the lacI repressor gene is deleted. Our strain also has gene deletions of araE and araFGH. LacY A177C is the only arabinose transporter in the cell allowing for better control of the P_BAD promoter and tunable repression by tCRISPRi.

A recent study by Peters et al. (2016) showed the power of CRISPR-based knockdown methods for studying essential genes in Bacillus subtilis. Their sgRNA libraries were cloned via inverse PCR, and dCas9 was under a xylose-inducible promoter. In contrast, our tCRISPRi system for E. coli uses one-step recombineering to make a tCRISPRi strain. The P_BAD promoter in the present work shows about 7.5% leaky expression, whereas the B. subtilis CRISPRi shows approximately 33% leakiness. Another important pioneering CRISPRi system was designed by the Marraffini group (2013), who used a plasmid-based system. We compare our tCRISPRi with these other two systems in Table 1. To see an example of applications of tCRISPRi to essential cell cycle genes, see Si et al. (2017).

Table 1. Comparison of different CRISPR interference system