These “off-target” genetic alterations demonstrate that certain CRISPR base editors need further refinement in a research finding of interest to pathologists

Could CRISPR DNA-editing technology unintentionally effect RNA as well? A new study conducted at Massachusetts General Hospital (MGH) suggests that it can. Clinical laboratories doing genetic testing will want to understand why this research implies that refinements to CRISPR may be needed for it to be accurate in therapeutic applications.

For years, a huge value of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) base editors have been their ability to edit genes or convert a specific DNA base without breaking the DNA. Now, the MGH scientists have discovered that certain CRISPR base editors may extend beyond the targeted DNA and perform unwanted edits to RNA, according to a news release.

“Most investigation of off-target base editing has focused on DNA, but we have found that this technology can induce large numbers of RNA alterations as well. This surprising finding suggests the need to look at more than just genetic alterations when considering unintended off-target effects of base editors in cells,” J. Keith Joung, MD, PhD, MGH Pathologist and Professor of Pathology at Harvard Medical School, stated in the news release.

The MGH scientists published their study in Nature.

How the MGH Researchers Found Off-Target Effects on RNA

The researchers had set their sights on developing a base editor that targets cytosine, according to the study.

“Previous studies of cytosine base editor specifically have identified off-target DNA edits in human cells. Here, we show that a cytosine base editor with rat APOBEC1 [rAPOBEC1] enzyme can cause extensive transcriptome-wide RNA cytosine deamination in human cells,” the scientists wrote in Nature.

According to the news release, when the researchers put base editors into human liver and kidney cells, they found their technology induced efficient edits at the target DNA site. However, they also discovered tens of thousands of cytosine-to-uracil edits in the cells. They found that deaminases, an enzyme that acts as a catalyst, which they used in their base editor to change DNA, also altered the RNA in the cells, Science reported.

“Base editors are still incredibly powerful tools. This is just another parameter we need to understand,” J. Keith Joung, MD, PhD (above), MGH Pathologist and Professor of Pathology at Harvard Medical School, told Science. (Photo copyright: Massachusetts General Hospital.)

The researchers developed a way to reduce the unwanted RNA edits, while maintaining the targeted DNA effects. They came up with cytosine base editor variants, which they dubbed SElective Curbing of Unwanted RNA Editing (SECURE).

“We engineered two cytosine base editor variants bearing rAPOBEC1 mutations that substantially decreased the number of RNA edits in human cells,” the researchers wrote in their study.

However, they also called for changes to how base editors are used. “For research applications, scientists using base editors will need to account for potential RNA off-target effects in their experiments,” the MGH news release notes. “For therapeutic applications, our results further argue for limiting the duration of base-editor expression to the shortest length of time possible and the importance of minimizing and accounting for potential impacts of these effects in safety assessments.”

 Other Studies Explore CRISPR

Other studies published earlier this year on mice and on rice also suggested that “modified CRISPR-Cas9 technology will need to be further refined before it can safely be used for research and therapeutic applications,” The Scientist reported.

Clinical laboratory leaders and pathologists recognize CRISPR technology is changing the way research is done for diagnosing disease as well as guiding treatment. Dark Daily has reported on key CRISPR developments over many years.

And now, though the MGH study may appear to be a set-back for CRISPR, it also may propel further research into possible therapeutic applications of CRISPR base editing. It’s a development worth watching.

—Donna Marie Pocius

Related Information:

CRISPR Base Editors Can Induce Wide-Ranging Off-Target RNA Edits

Transcriptome-wide Off-Target RNA Editing Induced by CRISPR-Guided DNA Base Editors

What Are the Key Differences Between DNA and RNA?

Powerful CRISPR Cousin Accidentally Mutates RNA While Editing DNA Target

Cytosine Base Editor Generates Substantial Off-Target Single Nucleotide Variants in Mouse Embryos

Cytosine, But Not Adenine, Base Editors Induce Genome-Wide Off-Target Mutations in Rice

Base Editors Cause Off-Target Mutations in RNA

CRISPR Base Editing, Known for Precision, Hits a Snag with Off-Target Mutations