Guide RNA truncation as a tunable approach for modulating base editing outcomes

Abstract

Base editing enables programmable nucleotide conversion but can be limited by bystander editing and unintended genomic alterations. Here, we investigated guide RNA (gRNA) truncation as a strategy to modulate base editing outcomes. On editing across multiple genomic loci using ABE8e in HUDEP-2 cells, we found that truncated gRNAs retained measurable editing activity at lengths as short as 15 nucleotides, although activity depended strongly on target sequence, gRNA length, and delivery method. Importantly, truncation altered editing efficiencies at individual nucleotides within the editing window, enabling selective reduction of bystander editing while preserving on-target conversion at several loci, including a therapeutically relevant HbE target. Truncated gRNAs also eliminated detectable large deletions at homologous globin loci, consistent with loss of Cas9 nickase activity at short gRNAs lengths. Similar length dependent effects were observed with cytosine base editing, PAM relaxed ABE8e, and in primary hematopoietic stem and progenitor cells (HSPCs), although the optimal gRNA length varied between contexts. Together, these findings establish gRNA length as an additional parameter for tuning nucleotide level base editing outcomes and reducing bystander editing.