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New CRISPR Base Editor Significantly Reduces DNA, RNA Off-Target Mutations

Gene Editing Generic

NEW YORK – Researchers in China have developed a more precise CRISPR base editing system that induces efficient editing with only background levels of genome-wide and transcriptome-wide off-target mutations.

Adenine base editors (ABEs) and cytosine base editors (CBEs) are known to efficiently and precisely edit point mutations in DNA with minimal off-target DNA editing. However, several studies have also shown that base editors may create unexpected problems by causing extensive transcriptome-wide off-target RNA editing in human cells, generating low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs, or inducing SNVs with frequencies more than 20-fold higher than the spontaneous mutation rate, for example.

CBEs and ABEs fuse the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases (CDAs), or in vitro evolved adenosine deaminases with CRISPR-Cas. However, studies showed that the originally reported BE3 CBE induced genome-wide off-target mutations in vivo, and could induce off-target mutations in transcriptomic RNA because the rat APOBEC1 moiety of BE3 can deaminate both deoxycytidine in DNA and cytidine in RNA.

In their new paper published on Monday in Nature Cell Biology, the Chinese researchers described what they called a transformer BE (tBE) system, which they said could induce precise base edits while eliminating genome-wide and transcriptome-wide off-target mutations. The tBE is fused to a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, which keeps the editor inactive at off-target sites. When binding to on-target sites, the tBE cleaves off the dCDI domain and catalyzes targeted deamination for precise base editing, the researchers said.

In an experiment in mice, the tBE created a premature stop codon in the Pcsk9 gene, significantly reducing serum levels of the PCSK9 protein and resulting in a 30 percent to 40 percent reduction in the animals' total cholesterol levels.

"The development of tBE establishes a highly specific base editing system, and its in vivo efficacy has potential for therapeutic applications," the authors wrote.

They began by quantitatively evaluating mutations at single-stranded off-target (OTss) sites and confirming that the APOBEC moiety of BEs could induce mutations in an sgRNA-independent manner. The investigators further determined the role of each CDA domain of the APOBECs containing dual CDA domains, and found that some inactive CDA domains exhibited dCDI activities. By linking a cleavable dCDI domain to the APOBEC moiety of BE, therefore, they set up a tBE system that remains inactive after being produced to avoid mutations at both sgRNA-independent OTss sites and the off-target sites that have sequences similar to on-target sites. (sgRNA-dependent OT sites).

Once the tBE binds at an on-target site, the dCDI domain is removed and the system is then activated for targeted deamination and editing. When they subsequently conducted whole-genome-wide and transcriptome-wide analyses, the researchers found that tBE induced no observable off-target mutations, even when correcting pathogenic mutations.

Overall, they said, the study demonstrated the potential of tBE in cell and gene therapies that require precise genome editing in primary or stem cells. They found that the width of the tBE editing window was around seven base pairs, which was relatively bigger than previously reported BEs, making it suitable for generating premature stop codons.

In the future, they added, tBEs could be engineered to have narrowed editing windows for more precise base editing. The system could also be combined with various orthologs of Cas9 to expand its targeting scope. And since there are many members of the APOBEC family, other dCDIs could be identified in the future that could also be added to the tBE system.