An essential gene is one that a prokaryote, such as a bacterium, requires to survive or reproduce. The challenge in studying these genes lies in a paradox: to understand a gene’s function, scientists often disrupt it and observe the consequences. If a gene is essential, any mutation that destroys its function will cause the cell to die, leaving no organism to study. This lethal outcome prevents a straightforward analysis and makes it difficult to confirm the gene’s role. To overcome this, researchers have developed specialized methods to study essential genes. These techniques conditionally control a gene’s activity, allowing scientists to keep the organism alive during initial manipulation and then induce the lethal effect at a chosen time.
The Challenge of Identifying Essential Genes
One method for studying gene function is targeted gene deletion through homologous recombination. Scientists introduce engineered DNA into a cell containing sequences that match the areas flanking the target gene, along with a marker like antibiotic resistance. The cell’s machinery can swap the original gene with the engineered DNA, creating a “knockout” mutant identifiable by its new resistance.
This approach works for non-essential genes because the resulting mutants survive and can be studied. When applied to a suspected essential gene, however, the technique fails to produce viable colonies. Replacing an indispensable gene is a lethal event, so no cells survive the procedure.
This absence of growth is only negative evidence. The experiment could have failed for other technical reasons, making it difficult to distinguish between an essential gene and an experimental error. This ambiguity necessitates methods that provide more direct proof of a gene’s function.
Conditional Mutagenesis Strategies
To overcome the limits of direct gene deletion, researchers use conditional mutagenesis. This approach allows a gene to function normally under permissive conditions, keeping the cell alive for lab cultivation. By shifting the organism to restrictive conditions, the gene’s function is turned off, leading to cell death and providing direct proof of its importance.
One strategy involves creating temperature-sensitive alleles. A mutation is introduced that makes the gene’s protein stable at a lower, permissive temperature. When the temperature is raised to a restrictive level, the protein misfolds and loses function, showing its necessity for survival.
Another method places the essential gene under the control of an inducible promoter. Promoters are DNA sequences that start gene transcription, and an inducible promoter is only active when a specific chemical, an inducer, is present. Scientists can engineer the prokaryote so the essential gene is controlled by this promoter. The cells are grown with the inducer, which can then be removed to shut off the gene and confirm its role.
Transposon-Based Mutagenesis for Genome-Wide Discovery
A method for discovering essential genes across an entire genome relies on transposons. Transposons, or “jumping genes,” are DNA segments that can move from one location in a chromosome to another. Scientists use this to create large libraries of random mutants by introducing a transposon into a population of bacteria. This generates millions of cells, each with the transposon inserted into a different location, disrupting the local gene.
The principle of this technique is a negative screen. After generating random mutations, the cell population is grown in the lab. If a transposon inserts into an essential gene, that cell cannot survive and is eliminated from the population. When researchers analyze the genomes of the survivors, they find transposon insertions throughout the chromosome except in the essential genes. These regions without insertions appear as “gaps,” indicating which genes are required for life.
This process has been refined into a technique called Transposon Sequencing (Tn-seq). This approach combines transposon mutagenesis with DNA sequencing to map the precise insertion site of every transposon in all surviving mutants. By comparing these insertion sites to the genome sequence, scientists can create a detailed, genome-wide map of gene essentiality, providing a list of genes required for survival under the tested conditions.
Targeted Gene Inactivation with CRISPRi
CRISPR interference (CRISPRi) offers a targeted way to study essential genes, contrasting with the random nature of transposons. Unlike the standard CRISPR-Cas9 system that cuts DNA, CRISPRi uses a deactivated Cas9 protein (dCas9). This dCas9 protein cannot slice DNA but can be guided to a specific gene by a guide RNA (gRNA). When the dCas9-gRNA complex binds to a gene’s starting point, it acts as a roadblock, preventing the cell from transcribing the gene.
This method silences or “knocks down” a specific gene’s expression without altering the DNA sequence. The utility of CRISPRi is enhanced by combining it with conditional systems. Researchers can place the CRISPRi components under the control of an inducible promoter, as described earlier. This allows them to grow cells normally, then add an inducer to activate dCas9 and silence the target gene on command.
Inducing gene knockdown at a chosen time is useful for validating if a gene, such as one identified by Tn-seq, is essential. Observing that cells stop growing or die only after the CRISPRi system is activated provides direct evidence of the gene’s function. While useful for testing individual genes, this system can also be scaled up with large libraries of guide RNAs to screen an entire genome.