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

Key findings

  • CRISPR-Cas base editors, a new technology for editing DNA, are able to change a single nucleotide without inducing breaks in double-stranded DNA
  • Researchers at the Massachusetts General Hospital Cancer Center found that base editors can induce tens of thousands of edits in the RNA of human cells
  • The researchers engineered two variant base editors and demonstrated in human cells that their use is associated with a 390- to 3800-fold reduction in the number of RNA edits

The standard CRISPR approach to gene editing relies on using an enzyme called Cas9 to induce targeted breaks in double-stranded DNA. More recently, fusion proteins called base editors have been developed to edit DNA even more precisely.

Base editors contain an enzyme called a deaminase that produces a specific alteration in DNA, enabling changes such as cytosine to thymine. But J. Keith Joung, MD, PhD, of the Center for Cancer Research at the Massachusetts General Hospital Cancer Center, and colleagues have found that cytosine base editors and the even newer adenine base editors can induce extensive off-target RNA edits in human cells.

In a letter published in Nature, they describe two base editor variants they've engineered that substantially decrease this phenomenon.

The Magnitude of Off-Target RNA Editing

The researchers tested the most commonly used cytosine-to-thymine editor in liver and embryonic kidney cancer cell lines. This editor induced efficient edits at the target DNA site, but it also led to thousands of cytosine-to-uracil edits throughout the transcriptome. The results were similar when the team tested an adenine-targeting base editor.

Addressing Unwanted RNA Off-Target Editing

To attempt to minimize unwanted RNA edits, the research team screened 16 editor variants that contained engineered deaminases. Two of these editors maintained the DNA editing efficiency of the original but substantially reduced the number of transcriptome-wide cytosine-to-uracil edits: by 390-fold for one editor and by 3800-fold for the other.

The researchers named these "selective curbing of unwanted RNA editing (SECURE)" base editor variants.

Potential Implications for Therapeutics

Scientists have been exploring potential therapeutic applications for CRISPR base editors, but the new findings suggest the duration and level of base-editor expression should be kept as short as necessary to minimize off-target RNA edits. In addition, the results suggest that investigators may wish to account for potential unwanted RNA edits when assessing base editor off-target profiles.

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