CRISPR-Cas9 connection to cancer prompts research to investigate different approaches to gene editing

Dark Daily has covered CRISPR-Cas9 many times in previous e-briefings. Since its discovery, CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, has been at the root of astonishing breakthroughs in genetic research. It appears to fulfill precision medicine goals for patients with conditions caused by genetic mutations and has anatomic pathologists, along with the entire scientific world, abuzz with the possibilities such a tool could bring to diagnostic medicine.

All of this research has contributed to a deeper understanding of how cells function. However, as is often the case with new technologies, unforeseen and problematic questions also have arisen.

CRISPR-Cas9 Connection to Cancer

Research conducted at the Wellcome Sanger Institute in the United Kingdom (UK) and published in Nature Biotechnology, examined potential damage caused by CRISPR-Cas9 editing.

“Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors, and a human differentiated cell line,” wrote the authors in their introduction.

Another study, this one conducted by biomedical researches at Cambridge, Mass., and published in Nature, describes possible toxicity caused by Cas9.

“Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR-Cas9 for genome engineering and screening in hPSCs [human pluripotent stem cells]. Moreover, as hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR-Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.”

Essentially what both groups of researchers found is that CRISPR-Cas9 cuts through the double helix of DNA, which the cell responds to as it would any injury. A gene called p53 then directs a cellular “first-aid kit” to the “injury” site that either initiates self-destruction of the cell or repairs the DNA.

Therefore, in some instances, CRISPR-Cas9 is inefficient because the repaired cells continue to function. And, the repair process involves the p53 gene. P53 mutations have been implicated in ovarian, colorectal, lung, pancreatic, stomach, liver, and breast cancers.

Though important, some experts are downplaying the significance of the findings.

Erik Sontheimer, PhD (above), Professor, RNA Therapeutics Institute, at the University of Massachusetts Medical School, told Scientific American that the two studies are important, but not show-stoppers. “This is something that bears paying attention to, but I don’t think it’s a deal-breaker,” he said. (Photo copyright: University of Massachusetts.)

“It’s something we need to pay attention to, especially as CRISPR expands to more diseases. We need to do the work and make sure edited cells returned to patients don’t become cancerous,” Sam Kulkarni, PhD, CEO of CRISPR Therapeutics, told Scientific American.

Both studies are preliminary. The implications, however, is in how genes that have become corrupted are used.

“It is unclear if the findings translate into cells actually used in clinical studies,” Bernhard Schmierer, PhD, co-author of a paper titled, “CRISPR-Cas9 Genome Editing Induces a p53-mediated DNA Damage Response,” told Scientific American.

Nevertheless, the cancer-cat is out of the bag.

Targeting RNA Instead of DNA with CRISPR-Cas13d

A team from the Salk Institute may have found a solution. They are investigating a different enzyme—Cas13d—which, in conjunction with CRISPR would target RNA rather than DNA. “DNA is constant, but what’s always changing are the RNA messages that are copied from the DNA. Being able to modulate those messages by directly controlling the RNA has important implications for influencing a cell’s fate,” Silvana Konermann, PhD, a Howard Hughes Medical Institute (HHMI) Hanna Gray Fellow and member of the research team at Salk, said in a news release.

The Salk team published their findings in the journal Cell. The paper describes how “scientists from the Salk Institute are reporting for the first time the detailed molecular structure of CRISPR-Cas13d, a promising enzyme for emerging RNA-editing technology. They were able to visualize the enzyme thanks to cryo-electron microscopy (cryo-EM), a cutting-edge technology that enables researchers to capture the structure of complex molecules in unprecedented detail.”

The researchers think that CRISPR-Cas13d may be a way to make the process of gene editing more effective and allow for new strategies to emerge. Much like how CRISPR-Cas9 led to research into recording a cell’s history and to tools like SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), a new diagnostic tool that works with CRISPR and changed clinical laboratory diagnostics in a foundational way.

Dark Daily reported on this breakthrough last year. (See, “CRISPR-Related Tool Set to Fundamentally Change Clinical Laboratory Diagnostics, Especially in Rural and Remote Locations,” August 4, 2017.)

Each discovery will lead to more branches of inquiry and, hopefully, someday it will be possible to cure conditions like sickle cell anemia, dementia, and cystic fibrosis. Given the high expectations that CRISPR and related technologies can eventually be used to treat patients, pathologists and medical laboratory professionals will want to stay informed about future developments.

—Dava Stewart

Related Information:

Repair of Double-Strand Breaks Induced by CRISPR-Cas9 Leads to Large Deletions and Complex Rearrangements

P53 Inhibits CRISPR-Cas9 Engineering in Human Pluripotent Stem Cells

CRISPR-Edited Cells Linked to Cancer Risk in 2 Studies

CRISPR-Cas9 Genome Editing Induces a p53-Mediated DNA Damage Response

Decoding the Structure of an RNA-Based CRISPR System

Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d

CRISPR Timeline

What Are Genome Editing and CRISPR-Cas9?

Federal Court Sides with Broad in CRISPR Patent Dispute

Top Biologists Call for Moratorium on Use of CRISPR Gene Editing Tool for Clinical Purposes Because of Concerns about Unresolved Ethical Issues

CRISPR-Related Tool Set to Fundamentally Change Clinical Laboratory Diagnostics, Especially in Rural and Remote Locations

Researchers at Several Top Universities Unveil CRISPR-Based Diagnostics That Show Great Promise for Clinical Laboratories