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Screening with CRISPR-Cas: the new life of the molecular scalpel

In 2012, scientists Jennifer Doudna and Emmanuelle Charpentier published an article in Science discussing the possibility of using the CRISPR-Cas system to edit the genome. Their work, supported by Feng Zhang’s later studies, showed that DNA could be quickly, efficiently safely modified thanks to a number of tools discovered in different types of microorganisms, including bacteria and archaea, at the end of the 1980's.

Since then, biotechnology has seen exponential growth in the research conducted on CRISPR-Cas, a system that works as an adaptive immunity mechanism for bacteria to resist the attack by virus, which can be applied to edit the genome, in such diverse areas as medicine, agriculture or food products.

This is not the only application of this microbial tool, also known as a ‘molecular scalpel’ thanks to the great precision it has shown in recent years. Scientists have worked to use CRISPR-Cas as a medical diagnosis method, with special attention to the recent studies published by the groups of Zhang and Doudna in Science. Now this work is enhanced by the possibility to use the system as a screening method.

Genome editing with CRISPR-Cas
​Source: Jennifer Doudna / University of California (Berkeley)

Understanding the toxicity of paraquat

A recent article published in the journal Nature Chemical Biology has shown the worth of CRISPR-Cas “to identify metabolic genes essential for paraquat-induced cell death.” Paraquat is a highly toxic herbicide, the use of which in agriculture is limited to the United States and prohibited in the European Union. For decades, the bipyridyl compound was used to control weeds, undergrowth and stubble, given its characteristics of rapid action on plants and deactivation in contact with the soil, according to an explanation given by the Autonomous University of Barcelona.

For humans, doses from 10 to 50 milliliters of the commercial concentrate are lethal if ingested orally. This polysystemic toxin especially impacts the lungs, where it accumulates in concentrations ten times higher than those seen in plasma, according to a study published in Critical Reviews of Toxicology. The main toxicity mechanism for this organ occurs due to the generation of free radicals that rapidly oxidize lung tissue.

Source: Pixabay

To understand the harmful effects of paraquat, researchers from the Feinberg School of Medicine, at Northwestern University in Chicago, used the CRISPR-Cas system to determine which genes could be associated with the toxicity of this herbicide in the body. To do this, they carried out a positive selection of the genes “whose loss allowed cell survival in the presence of 110 μM PQ, a concentration of PQ that greatly decreases cell viability.”

This way, they were able to determine three genes key to paraquat-induced cell death: POR, ATP7A and SLC45A4. Navdeep Chandel's group demonstrated that cells that did not have these genetic sequences were resistant to lethal concentrations of the herbicide. Individuals with genetic mutations in these regions could be susceptible to paraquat poisoning if they worked in contact with the herbicide (for example, on a farm), claim the authors of the study. “The biology of oxidative stress is still a mystery. CRISPR positive-selection screens could be a way to figure it out,” states Chandel.

​Source: Benjah-bmm27 (Wikimedia)

On another note, American scientists also conducted a CRISPR-based screening to carry out a negative selection of those genes that sensitize cells of the Jurkat line when faced with low paraquat concentrations (25 μM). That is how they determined the importance of genes SLC31A1, which encodes the plasma-membrane-bound CTR1 copper importer protein, and SOD1, which encodes the copper and zinc-dependent cytoplasmic antioxidant enzyme SOD1.  “Now, we can go in and test how agents of oxidative stress work. The beauty of the paper is in the power of these unbiased genetic screens we can now use with CRISPR technology”, states the Principal Investigator.

​Although interest in CRISPR-Cas has focused in recent years on genomic editing, with potential applications in medicine as a possible genetic therapy against a number of diseases, the truth is that its use does not stop there. This work, together with articles like the one recently published in Methods in Molecular Biology show the possibility of combining CRISPR-Cas with technologies such as DNA sequencing to be able to identify the function of certain genes of interest in various biological processes “in a high-throughput fashion.” The specificity and efficiency of this molecular tool as compared to other techniques like interference RNA has already been used, for example, to determine mutations that promote cancer treatment resistance or in the development of new drugs.