CRISPRi functional genomics in bacteria and its application to medical and industrial research

Abstract

SUMMARYFunctional genomics is the use of systematic gene perturbation approaches to determine the contributions of genes under conditions of interest. Although functional genomic strategies have been used in bacteria for decades, recent studies have taken advantage of CRISPR (clustered regularly interspaced short palindromic repeats) technologies, such as CRISPRi (CRISPR interference), that are capable of precisely modulating expression of all genes in the genome. Here, we discuss and review the use of CRISPRi and related technologies for bacterial functional genomics. We discuss the strengths and weaknesses of CRISPRi as well as design considerations for CRISPRi genetic screens. We also review examples of how CRISPRi screens have defined relevant genetic targets for medical and industrial applications. Finally, we outline a few of the many possible directions that could be pursued using CRISPR-based functional genomics in bacteria. Our view is that the most exciting screens and discoveries are yet to come.

Keywords: CRISPR-Cas9; ESKAPE pathogens; antibiotics; biofuels; bioproducts; genetic screens.

Fig 1

Mechanism of gene knockdown by CRISPRi. An sgRNA binds to dead dCas9 and directs the complex to a complementary DNA target sequence (protospacer) with an adjacent PAM where the complex physically blocks elongation by RNAP. Asterisks in dCas9 mark point mutations which inactivate nucleolytic activity. sgRNA designs that target the non-template strand are generally more effective for CRISPRi knockdowns.

Fig 2

Graphic overview of a CRISPRi functional genomics screen and analysis for a library of k strains in n competition assays. Oligo libraries can be amplified and cloned into delivery vectors to generate knockdown strain libraries. Phenotyping typically consists of growth in a pooled, competitive environment in which some strains increase in frequency (increased relative fitness) while others are depleted from the pool (decreased relative fitness). Amplicons of sgRNA spacers act as barcodes to identify and count each knockdown strain. Once counted, information about the fitness of knockdown strains can be analyzed and modeled.

Fig 3

Schematic depicting medical or industrial and environmental applications for chemical genomics using CRISPRi. In this representation, “target” is the gene product or pathway that is directly inhibited by the antimicrobial or toxin. Once identified, targets may be leveraged in further basic and/or applied research.

Fig 4

Examples of gene targets with (A) medical or (B) industrial and environmental relevance which may be identified and/or characterized with CRISPRi. Blunt arrows depict gene knockdown by CRISPRi (red) or chemical-mediated inhibition (black). Pointed arrows depict specific outcomes (black).