California researchers use CRISPRi to devise new method for studying essential genes

Although essential genes are only a tiny part of the genes of most organisms (about 5%-10%), they are important because they are responsible for much of the organism's protein synthesis and because they are the targets of most antibiotics. Essential genes are notoriously hard to study, however, because their removal leads to the death of the organism.

That situation has changed. 

A group of scientists at the University of California San Francisco and at Stanford has devised a method for decreasing the production of essential genes without removing them from the organism. The researchers have created a new resource for scientists studying essential genes, a comprehensive library of hundreds of CRISPRi strains, each of which can deplete an essential gene of the well-studied bacterium Escherichia coli.

Their work appeared in the journal of the American Society for Microbiology, mBio, September-October 2021. 

The new study describes the design and construction of the library in E. coli. It also profiles the morphological and growth phenotypes of the library, including some previously unrecognized aspects of bacterial physiology. One of the new discoveries the group made is that depleting different essential genes affects the reproductive rate of the bacteria differently.

The library will be available to microbiologists through the Coli Genetic Stock Center for research.

CRISPRi can help develop new therapies

CRISPR interference (CRISPRi), an offshoot of the CRISPR technology used for the precise editing of a genome in vivo, allows varying degrees of inhibition of the expression of any gene. This study is the first time an arrayed library has been constructed to target the essential genes of any Gram-negative bacteria. Gram-negative bacteria can cause serious infections, and because they have a hardy cell envelope, many antibiotics cannot penetrate into the cell, making Gram-negative cells inherently resistant to many antibiotics.

Co-author Horia Todor, a specialist in microbiology and immunology at the University of California School of Medicine in San Francisco, described the importance of the work: 

"Our library will help the scientific community investigate the function of essential genes in E. coli, and will potentially serve as an important screening tool for new antibiotics," she said. "Because E. coli serves as a model organism for Gram-negative bacteria, a broad category that includes pathogens such as Pseudomonas aeruginosa, Salmonella enterica, and Acinetobacter baumanni, this work will help us develop new drugs and therapies to fight these pathogenic bacteria."

The strain library, Todor said, "consists of hundreds of CRISPRi strains each of which turns off a specific essential gene. This library is arrayed in a grid, so we can study all or just some of the strains."

Antibiotics target essential genes

“E. coli has about 4,000 genes, but only about 300 are essential," Todor noted. "As you might expect, these 300 essential genes are at the core of most cellular pathways (e.g. translation, transcription, cell wall synthesis, DNA replication, etc.) and are the primary targets of almost all antibiotics." 

Todor demonstrated how CRISPRi improves the usual method for studying essential genes.

"The way we generally study genes is by deleting them and observing how the organism changes," he said. "For example, if we delete a gene and E. coli can no longer metabolize galactose, we know that the gene we deleted is involved in galactose utilization. Similarly, if we delete a gene and E. coli goes from rod-shaped to round, we know that the gene we deleted is important for maintaining cell shape.

"This approach doesn't work for essential genes," he added. "By definition, deleting an essential gene kills the cell. CRISPRi allows us to study essential genes by lowering their expression enough to affect the cells but not enough to kill them. Moreover, the CRISPRi system can be turned up or down, allowing us to see how cells respond to losing a bit or a lot of an essential gene."

Creating the library

Todor described how the researchers created the strain library and studied different strains.

"To perform an initial characterization of our library, we looked at how well each of our strains grew, as well as how lowering the level of each essential gene affected the shape of the cells," he said. "We found that cells responded differently when we reduced the expression of different essential genes. First, we found that lowering the expression of some genes affected cell growth much more than lowering the expression of others, suggesting certain genes would be better drug targets than others."

"Second, we found that cells responded to lowered expression of certain essential genes by activating stress pathways, such as the SOS response and the stringent pathway," he added. "Finally, we found that some essential genes, such as mreB, have built-in feedback mechanisms that fight the CRISPRi system to maintain cellular viability." 

The promise for research

Todor elaborated on the implications of the team's finding that when translation was slowed, that it causes an extended lag phase in the bacteria.

"Translation, which is the process of making more proteins, is often associated with rapid cellular growth," he said. "Our finding that slowing translation causes an extended lag phase when transitioning from stationary phase to exponential growth suggests that translation is important not only for fast growth, but also for making physiological and metabolic transitions, such as exiting stationary phase."

Even a slight "knockdown of essential ribosomal proteins is sufficient to substantially lengthen the lag phase," the researchers state. They found that in this period the bacterial cells grew linearly, rather than exponentially.

The study concludes that the use of CRISPRi libraries for Gram-positive bacteria has already contributed to "our understanding of essential gene function and the interplay between essential processes. We anticipate that the E. coli library described here will set the stage for similarly powerful advances in Gram-negative bacteria," it concluded.

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Melanie R. Silvis, et al. "Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli," mBio, September-October 2021. https://journals.asm.org/doi/pdf/10.1128/mBio.02561-21