Everett Weinstein Intern GEN

Fusion of CRISPR and APEX2 Stands to Bolster Genome Mapping Capabilities

Researchers from the Broad Institute invented a new technique for isolating DNA-associated proteins which, when perfected, may eventually replace the most commonly used current method, chromatin immunoprecipitation (ChIP).

The team created a new protein called Caspex designed for affinity tagging proteins at specific points in the human genome. This new tool is part of the team’s development of genomic locus proteomics, also known as GLoPro.

Chromatin immunoprecipitation has long been utilized for discerning the relationship between proteins and DNA, and was used specifically to determine where certain proteins were located along our genome. ChIP has a few limitations, including the requirement for a list of suspect proteins and the need for specific and high affinity antibodies for every protein one wants to test. As Samuel A. Myers, PhD, a research scientist at the Broad Institute of MIT and Harvard, stated, “ChIP has been super powerful, but you need to know what you’re looking for and you need to have good reagents for it.”

ChIP is consequently a little too convoluted to use effectively for discovery applications, leaving obvious room for improvement. GLoPro picks up the slack where ChIP falls short as Caspex identifies proteins within specific, nonrepetitive, genomic loci, absent of the requirement for genomic augmentation according to the researchers.

 “What this allows you to do is to unbiasedly and without any prior knowledge identify proteins that are associated with this particular locus that you’re interested in,” says Dr. Myers. “You can start discovering things that were there without having any idea of what was there prior.”

Crux of the Experiment

A catalytically inactive Cas9 enzyme, known as dCas9, was fused with a peroxidase, APEX2, to form the crux of this experiment: Caspex. CRISPR-Cas9 is famous for its ability to target specific sequences in our genetic code out of our roughly 3 billion base pairs.

“We use the easily reprogrammable nature of Cas9 to tile the target locus, because I think its accuracy is up for debate,” says Dr. Myers.

The APEX2 enzyme tags proteins within a certain area by biotinylating them—or attaching a biotin moiety—in the presence of hydrogen peroxide. According to the team, they “chose APEX2 for its small labeling radius and short reaction time.”

These two proteins synergize extraordinarily well, and when combined the resulting Caspex carries both the compact marking properties of APEX2 and the targeting capabilities of CRISPR.

During an experiment testing the viability of the Caspex protein, the scientists wrote that Caspex “biotinylated proteins within approximately 400bp of either side of its targeted locus.” Taking into consideration the size of the human genome, that’s a feat that would result in the isolation of an incredibly small portion of our DNA and provides a precise net for honing in on any sequence of interest. The biotinylated proteins can then be enriched and analyzed for further proteomic investigation.

While the field of genomics is growing rapidly, there are still many who cling onto a negative stigma surrounding ideals of genetic augmentation. Many of the criticisms are derived from the claims that the implementation of synthetic DNA is unethical, and that the splicing of the genome can result in a myriad of unintended side effects.

However, with the advent of Caspex acting as a tool that can be used to enhance our ability to locate proteins at specific promoters, scientists can delve deeper into the possibility of using epigenetics to amend certain genetic determinants without the need for more aggressive genomic changes.

“I don't think this method has much to do with altering the genome,” notes Dr. Myers, “It has more to do with describing and expanding the mechanisms of how the genome is working.”

Future Potential

The key words here are that GLoPro doesn’t alter the genome. However the future potential to adjust the expression of the genome is certainly a possibility. “We could use Caspex to identify which DNA elements and associated proteins—potentially druggable targets—actually control the expression of the genes one’s looking at. We can then combine this with analyzing signaling pathways to better understand what’s happening to actually enact changes in gene expression,” continues Dr. Myers.

With regards as to what the immediate future has in store for GLoPro, Dr. Myers explains that “short term, the next thing that I want to start looking at is before and after conditions, so what’s the first protein to show up shutting off a gene during differentiation, or what's the first proteins to show up during gene activation?”

This new insight granted by GLoPro on the location of certain DNA-associated proteins, or clusters of proteins, could result in a vast array of possibilities for the subtler side of genetic manipulation in epigenetics. The ability to incite change—not by modifying the human genome but instead by modifying the production of specific proteins through transcription control—could allow for less invasive adjustments to be made without generating as many potential consequences.

The beauty of epigenetics is that it explicitly alters the phenotype but doesn’t make any changes to the genotype. Caspex could be vital in ameliorating our capabilities within this field. But first and foremost it stands to become a valuable asset as a cartographer of genomic mechanisms.

The entire article can be found through Nature Methods scientific journal.


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