Abstract
We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties.
Keywords: CRISPR interference, Gene expression, Gene knockdown, Recombineering
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 PBAD 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 PBAD 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
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Images were acquired at 100x magnifications using Nikon Ti-E microscope equipped with a Neo sCMOS camera (Andor) and Nikon NIS-Elements software. For the cell length measurements, phase contrast technique was used, and images have 2,560 x 2,160 resolution and 16 bit grayscale. The fluorescent images for msfGFP cells were obtained with illumination by an OBIS 488 nm laser from Coherent and the 59022 filter cube from Chroma. For each cell, the fluorescence signal was integrated and normalized by the projected area of the cell after background subtraction. Illumination across the field of view was homogeneous with less than 5% variations. For each experimental condition, we acquired 150-200 images containing at least 10,000 cells, and calculated the average value of each steady-state population for the data in Figure 1. We developed and used custom high-throughput image analysis software optimized for our experiments using Python and OpenCV library. For more detailed information on data analysis is available in Materials and Methods as well as Supplementary Information in Li et al., 2016 with specific examples of MreB, DnaA, RepA, SeqA knockdown in Si et al., 2017.
Recipes
Acknowledgments
We thank Don Court (NCI/NIH) for collaboration with the published work in Li et al. (2016). This work was supported by the Paul G. Allen Foundation, the Pew Charitable Trusts, the National Science Foundation CAREER Award, and NIH R01 GM118565-01 (to S.J.). We thank Sarah E. Cox for critical reading of the manuscript.
References
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