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. 2020 Nov 6;9:e60083. doi: 10.7554/eLife.60083

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© 2020, de Wet et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 1. — (A) An arrayed CRISPRi library was designed to target 294 essential M. smegmatis genes. For each gene, the highest efficiency sgRNA was identified from a previous pooled CRISPRi-Seq screen (de Wet et al., 2018) and synthesized as an annealed oligonucleotide. Cloning was performed at scale, followed by electroporation into an M. smegmatis ParB-mCherry reporter strain (Santi and McKinney, 2015). (B) A total of 276 sequence-verified transformants produced a greater than twofold decrease in colony size (Kritikos et al., 2017) when spotted on 7H10 agar containing the inducer, anhydrotetracycline (ATc) compared to the same cells spotted onto solid medium without ATc, confirming ATc-dependent growth inhibition.

Figure 1—figure supplement 1. — (A) An arrayed CRISPRi library was designed to target 294 essential M. smegmatis genes. For each gene, the highest efficiency sgRNA was identified from a previous pooled CRISPRi-Seq screen (de Wet et al., 2018) and synthesized as an annealed oligonucleotide. Cloning was performed at scale, followed by electroporation into an M. smegmatis ParB-mCherry reporter strain (Santi and McKinney, 2015). (B) A total of 276 sequence-verified transformants produced a greater than twofold decrease in colony size (Kritikos et al., 2017) when spotted on 7H10 agar containing the inducer, anhydrotetracycline (ATc) compared to the same cells spotted onto solid medium without ATc, confirming ATc-dependent growth inhibition.