Fig. 1: Optimizing dsODN Tagging for Enhanced Detection of CRISPR-Cas9 Off-Target Effects.

a OliTag-seq Overview: The diagram details the OliTag-seq methodology for detecting CRISPR-Cas9 off-target effects. Cells are electroporated with Cas9-sgRNA and dsODNs, and genomic DNA is sequenced after three days per the OliTag-seq protocol, pinpointing dsODN-tagged DSBs. b dsODN Sequences Comparison: This section juxtaposes the dsODN sequences from GUIDE-seq (34 nt) with OliTag-seq (39 nt), outlining structural features like 5′ phosphorylation (‘P’) and phosphorothioate bonds (‘*‘). Extended GC clamps on the 39 nt ODNs are highlighted, indicating their potential for improved performance. c dsODN Integration Efficiency: The efficiency of full-length dsODN integration at six genomic sites is compared for 34 nt and 39 nt ODNs (n = 3–4 per site). The frequency of reads with complete ODNs relative to total edits is calculated, with the 39 nt ODN efficiency normalized to the 34 nt ODN. d NHEJ-Mediated dsODN Integration and Small Molecules: This examines the effects of small molecules on NHEJ-mediated dsODN integration (n = 5), using M3814 as a negative control. Integration frequencies are normalized against controls to evaluate the potential enhancement by these treatments. e dsODN Tagging in K562 Cells: The analysis compares sequencing read counts for DSBs tagged with 34 nt and 39 nt dsODNs in K562 cells, following electroporation with Cas9 and specific sgRNAs. For balanced comparison, counts for 39 nt ODNs are normalized to 34 nt ODNs. f Off-Target Site Comparison: Off-target sites tagged by 39 nt and 34 nt ODNs in genes like TRAC, EMX1, and VEGF3 are compared (n = 4–8 per site). Normalized data for 39 nt ODNs are displayed as mean ± s.d., with statistical significance determined by paired two-tailed Student’s t tests and annotated with adjusted p-values.