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bioRxiv · 10.1101/358275

CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

de Wet, T. J.·Gobe, I.·Mhlanga, M. M.·Warner, D. F.
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Abstract

High-throughput essentiality screens have enabled genome-wide assessments of the genetic requirements for growth and survival of a variety of bacteria in different experimental models. The reliance in many of these studies on transposon (Tn)-based gene inactivation has, however, limited the ability to probe essential gene function or design targeted screens. We interrogated the potential of targeted, large-scale, pooled CRISPR interference (CRISPRi)-based screens to extend conventional Tn approaches in mycobacteria through the capacity for positionally regulable gene repression. Here, we report the utility of the \"CRISPRi-Seq\" method for targeted, pooled essentiality screening, confirming strong overlap with Tn-Seq datasets. In addition, we exploit this high-throughput approach to provide insight into CRISPRi functionality. By interrogating polar effects and combining image-based phenotyping with CRISPRi-mediated depletion of selected essential genes, we demonstrate that CRISPRi-Seq can functionally validate Transcriptional Units within operons. Together, these observations suggest the utility of CRISPRi-Seq to provide insights into (myco)bacterial gene regulation and expression on a genome-wide scale.

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de Wet, T. J., Gobe, I., Mhlanga, M. M., Warner, D. F.. 2018-06-29. CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units. https://doi.org/10.1101/358275

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