CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units
Year: 2018
Type: preprint
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.
DOI: 10.1101/358275
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: Chan Zuckerberg Initiative, South African Medical Research Council, Department of Science and Technology, Ministry of Science and Technology, India, Norges Forskningsråd, University of Cape Town, Medical Research Council, National Institute of Child Health and Human Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development
OpenAlex ID: W4249215296