Preprint: Guide RNA length changes which neighboring DNA bases a base editor modifies
Preprint: Guide RNA length changes which neighboring DNA bases a base editor modifies
On September 13, a preprint was published on how guide RNA length affects base editing in human cells. At a site in the beta-globin gene, a 15-nucleotide guide retained the intended substitution, while the neighboring substitution occurred at approximately 3% of its frequency. At a site in the gamma-globin gene, the minimum length that supported editing depended on the delivery method.
A guide RNA directs the Cas9 protein to a selected site in DNA. A base editor couples Cas9 to an enzyme that changes individual bases within a short, unwound stretch of DNA. If another base that the enzyme can modify lies next to the intended base within this stretch, both bases are modified.
The authors shortened guide RNAs from 20 to 10 nucleotides by removing nucleotides from one end and compared the results at several DNA sites. They tested whether this would alter the enzyme’s access to neighboring bases while keeping the editor itself unchanged.
At the beta-globin gene site, they targeted the HbE mutation. The intended substitution was at base A9, while a substitution at the neighboring A10 produced another hemoglobin variant, Hb Aubenas. The 15-nucleotide guide produced the highest frequency of A9 editing in this series, with a ratio of A10 to A9 substitutions of approximately 0.03.
At the gamma-globin gene site, the minimum guide length at which the authors observed editing depended on the delivery method. In HUDEP-2, a blood progenitor cell line engineered to continuously produce the editor, a 15-nucleotide RNA supported measurable editing. Following transient delivery of the editor and RNA into HUDEP-2 and primary blood progenitor cells, measurable editing at the same target required at least 17 nucleotides.
The similarity between globin genes poses another problem: Cas9 can target two sites at once. In a 2024 study by the same group, simultaneous nicks at such sites were associated with large deletions between them. In the current preprint, PCR assays designed to detect new DNA fragments formed by such a deletion did not detect the expected fragments with guides of 15–16 nucleotides. In the authors’ comparison, dABE8e, an editor variant whose Cas9 does not nick a DNA strand, achieved a higher editing frequency than ABE8e with a 16-nucleotide guide.
In these experiments, guide RNA length changed which substitutions occurred and how frequently they occurred. The outcome also depended on the target, editor, and delivery method.