CRISPR—short for ‘clustered regularly interspaced short palindromic repeats’—is a nobel-prize winning scientific advancement in genetic modification technology. It was initially developed by Dr. Jennifer Doudna of Harvard University, and is based on the naturally occurring gene-editing system found in bacteria. Researchers now use this new method to modify the DNA of various organisms, potentially being able to make advancements in disease treatment, improving resilience of crops, correcting genetic defects, and more.
To make an understatement, the introduction of CRISPR into the scientific community has been nothing short of groundbreaking, but researchers from Rice University have raised their own doubts about this seemingly miraculous technology, and whether or not it is as fool-proof as it’s presented to be. In response to this question, they have begun to lead an effort with a goal “to reveal potential threats to the efficacy and safety of therapies based on CRISPR-Cas9…even when it seems to be working as planned.”
CRISPR-Cas9 was designed to treat sickle-cell anemia. In order to combat this disease, the technology works to edit large sequences in a patient’s DNA, therefore aiming to change their DNA and erase the aspect of it that makes them suffer from the illness. However, researchers have begun to fear that taking such a large step as this (erasing large portions of one’s DNA) is presumptuous, and could possibly yield dangerous, long-term effects, since this genetic modification CRISPR allows will only further spread throughout the patient’s body through stem cell division/differentiation.
These fears mainly stem from the fact that scientists are not sure how DNA strands are able to rejoin after so many of their sequences have been cut out, and therefore, separated. However, bioengineer Gang Bao of Rice University has other concerns, as well: “large deletions (LDs) can reach to nearby genes and disrupt the expression of both the target gene and nearby genes.’”
Gene expression is a very complex process that occurs in the cells of all organisms, but which can be broken down into two major steps: transcription—”synthesis of RNA using information from DNA”—and translation—”synthesis of a polypeptide or protein using information in the mRNA.” This process running smoothly is extremely important, as the ‘information from the DNA,’ or amino acid bases, need to be copied exactly without any mistakes, duplicates of bases, etc..
Bao also expresses another concern about CRISPR-Cas9: “‘you could also have proteins that misfold, or or proteins with an extra domain because of large insertions. All kinds of things could happen, and the cells could die or have abnormal functions.’”
With so many hypotheses at play, Bao and his research team knew they had to somehow figure out answers: they developed a technique called SMRT—’single molecule, real time’—that utilizes molecular identifiers to seek out and find accidental LDs, long insertions, and chromosomal rearrangements that are located at a Cas9 cutting site. To do this, a machine was used called the ‘LongAmp-seq’ (long-amplicon sequencing) to emphasize the presence of particular DNA molecules. This allows for the quantification of LDs and large insertions on a DNA strand.
Researchers used streptococcus pyogenes as a medium. With this bacteria, they edited enhancers such as beta-globin (HBB), gamma-globin (HBG), and B-cell lymphoma/leukemia 11A (BCL11A), and genes such as PD-1 gene in T-cells of sickle-cell anemia patients.
In testing these, they found incredible results: across the 3 enhancers and 1 T-cell gene, the average frequency of several thousand large DNA deletions averaged a whopping 20.025%.
While it is unclear at this time whether Bao’s team’s discoveries will unveil consequences of genes modification by CRISPR technology, they state that they will work to “determine the biological consequences of gene modifications due to Cas9-induced double-strand breaks,” and look forward to testing if “‘these large deletions and insertions persist after the gene-edited HSPCs are [transplanted] into mice and patients.’