Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Reinforced CRISPR Interference (CRISPRi) Enables Reliable Multiplex Gene Repression In Phylogenetically Distant Bacteria

Authors:

Joshua R. Elmore1* (joshua.elmore@pnnl.gov), Ritu Shrestha1, Andrew Wilson1, Winston Anthony1, Elise Van Fossen1, Andrew Frank1, Ryan M. Francis1, Henri Baldino1, Jean Rivera1, Bhavya Gupta1, Jay Huenemann2, Valentine Trotter3, Adam Guss2, Adam Deustchbauer3, Robert G. Egbert1

Institutions:

1Pacific Northwest National Laboratory; 2Oak Ridge National Laboratory; 3Lawrence Berkeley National Laboratory

Goals

The Persistence Control Science Focus Area (PerCon SFA) at PNNL seeks to understand plant-microbiome interactions in bioenergy crops to establish plant-growth-promoting microbiomes that are contained to the rhizosphere of a target plant. This vision requires the discovery of exudate catabolism pathways from plant roots, the elimination of genes that support fitness in bulk soil environments without decreasing rhizosphere fitness, and the engineering of rhizosphere niche occupation traits in phylogenetically distant bacteria. Researchers anticipate the impacts of these efforts will be to increase understanding of plant-microbe interactions and to extend high-throughput systems and synthetic biology tools to non-model microbes.

Abstract

Persistence control is an engineering approach in which survival of genetically modified microorganisms is restricted to a target environmental niche. However, the current knowledge of gene functions is insufficient to rationally design persistence control traits. CRISPR interference uses the combination of a Cas protein and a guide RNA to repress gene expression in a sequence-specific manner and can be used to identify genes involved in growth and survival.

However, current CRISPRi tools each have substantial caveats that limit their application outside of simple, well-controlled laboratory conditions. The PerCon SFA is using the sorghum rhizosphere as a target environmental niche and has developed a multi-guide CRISPR interference (CRISPRi) system to simultaneously repress multiple genes in this complex environment. Researchers use Serine recombinase-Assisted Genome Engineering (SAGE) to simultaneously integrate the CRISPRi protein dCas12a and its guide array into the host chromosome (Fig. 1). Fine-tuning the expression each component is critical to maximize gene repression and minimize fitness defects. Unlike dCas9, the most common CRISPRi system, the primary CRISPR array transcript processed by dCas12a creates distinct guide RNAs, enabling researchers to repress multiple genes with a single transcriptional unit (Fig. 1). To address the unpredictable efficiency of single-guide repression, researchers show that reinforcing CRISPRi with multiple guides per gene greatly improves both the magnitude and reliability of gene repression for fluorescence and cell-growth gene targets. SAGE is an organism-agnostic toolkit that enables the creation of robust CRISPRi systems in bacteria from multiple phyla (Fig. 2).

We are currently using CRISPRi in the rhizobacteria Pseudomonas facilor and Pseudomonas fluorescens to prototype multi-gene deletions and perform high-throughput functional screens. Pooled multiplex CRISPRi screens enable researchers to evaluate whether pairs of genes that conditionally impact fitness are involved in the same or distinct physiological processes under abiotic stresses. Additionally, researchers have begun comparing the outcomes of genome-scale single guide CRISPRi, multiple guide CRISPRi, and randomly barcoded transposon mutant screens. Early results highlight advantages and weaknesses to each approach and have found that co-analysis of data from distinct genome-wide screens can identify proteins with independently functioning protein domains.

Image

Figure 1. Overview of how SAGE is applied for constructing a multiplex CRISPR interference system.

Fig. 1. Overview of how SAGE is applied for constructing a multiplex CRISPR interference system.

References

Elmore, et al. 2023. “High-Throughput Genetic Engineering of Nonmodel and Undomesticated Bacteria Via Iterative Site-Specific Genome Integration,” Science Advances 9(10), eade1285. DOI:10.1126/sciadv.ade1285.

Funding Information

This research was supported by the U.S. DOE, BER Program, as part of BER’s GSP, and is a contribution of the Pacific Northwest National Laboratory (PNNL) Secure Biosystems Design Science Focus Area “Persistence Control of Engineered Functions in Complex Soil Microbiomes”. PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RL01830.