A newly designed gene editing strategy

The power and convenience of modern word processing programs such as Microsoft Word have revolutionized our daily tasks. Need to create a quick resume for a new job opportunity? Are you procrastinating on tomorrow's final document? Even by creating a quick shopping list: most of us resort to word processing programs as guardians of our written lives. The functionality is impressive and unlike its archaic predecessor, the typewriter only allows a few keystrokes to the keyboard, which allows to modify, delete or add words at the request of the user.

For years, researchers have been looking for ways to mimic these capabilities, but at the genetic level, to accurately and efficiently modify genetic information. Developing a "Microsoft Word of DNA" would allow scientists to better study the functioning of individual genes and the mutations that contribute to genetic disorders. With a single discovery called CRISPR-Cas9, scientists switched from a typewriter to a DNA word processor; edit genes with the same ease, precision and versatility. Technology has gained popularity and popularity because of its flexibility and customizable character.

Although powerful, CRISPR is far from being a perfect technique and presents, like any other technique, its disadvantages and its limits. One of its main uses is to precisely target and edit a gene of interest so that it does not work in a specific type of cell. When a gene is rendered non-functional, any characteristic change in the cell (known as the phenotype) can be studied in order to get a better picture of what that particular gene does. But how CRISPR mutates individual genes can be a challenge for researchers. When placed in a cell, the CRISPR-Cas9 system accurately mutates a targeted gene by cutting the cell's DNA. The cell then repairs its broken DNA primarily through a process called non-homologous end junction (NHEJ). But this repair process is prone to errors and can cause variability in the repaired DNA; often leading to substitutions, deletions or additions to the genetic code. In addition, the effectiveness of CRISPR can vary, making both copies of a targeted gene nonfunctional or sometimes only one. These unknown changes in DNA caused by CRISPR can make it extremely difficult for scientists to interpret the underlying genetic cause of an observed phenotype, making the tool much less useful .

In a recent publication in Cell reports, the Taniguchi laboratory of the Max Planck Institute of Neuroscience (Florida) (MPFI) have developed a new methodology for linking phenotype to genotype. By innovatively combining the advanced technique of laser microdissection with cell genotyping, the Taniguchi laboratory has designed an experimental pipeline capable of studying the CRISPR-induced effects in cells while determining with accurately the exact modifications of the DNA that caused them. This new protocol will open new avenues of study in neurobiology and strengthen the already powerful capabilities of CRISPR.

"Although CRISPR specifically targets a gene of interest, because of NHEJ, its effects can be highly variable," says Andre Steinecke, Ph.D., researcher and first author of the publication. "CRISPR can leave cells with totally nonfunctional genes, weakened genes or even sometimes improve their function.This is not a problem if you delete one that causes a very visible effect in the cells because you can easily visualize the change and the absence of the protein But some, especially brain genes, have no obvious effects or are very difficult to visualize.Our goal was to create a broadly applicable strategy, able to determine reliably the exact genetic cause and correlate it with observed phenotype. "

To validate their strategy, the MPFI team designed the CRISPR technology to target a gene in pyramidal neurons encoding a critical structural protein called Ankyrin-G (AnkG). Normally, the AnkG protein is confined to a specialized region of the neuron called the initial segment of the axon (AIS), which is responsible for generating action potentials. When the AnkG is removed, the AIS undergoes a noticeable thickening that can be detected by microscopy. With this feature, neurons lacking AnkG could be easily distinguished and their exact genotype could then be confirmed. They discovered that, primarily, neurons transfected with their CRISPR probe exhibited AnkG loss as well as a substantially thickened AIS. However, a small portion of the CRISPR-transfected neurons still had AnkG levels and AIS thickness comparable to wild type neurons; demonstrating the variable effects of CRISPR on different cells.

To probe and confirm the underlying genetic causes, the team then used laser microdissection to isolate and extract individual neurons whose phenotype had already been characterized. Once extracted, the team sequenced each individual cell separately to determine the genotype. They found that their strategy could reliably and reproducibly link observed phenotypes to genotype, where AnkG-free neurons with thickened axons showed mutational loss mutations in both copies of the gene, whereas neurons with normal levels of AnkG did not show either neurons transfected with CRISPR) or normal genotypes (control neurons). The team then confirmed its strategy using two additional genes, MeCP2 and Satb2, concluding that their process could again effectively correlate the observed trait to the underlying genetics.

"CRISPR / Cas9-based gene targeting is very promising for the systematic understanding of the molecular bases underlying the assembly, function and dysfunction of neuronal circuits," notes Hiroki Taniguchi, Ph.D. between the genotypes determined by our unique cell sequencing and those deduced from the phenotype assessment, suggests that our approach is a powerful new method that can improve the reliability and extend the applications of CRISPR techniques. "