Optimized CRISPR/Cas9 Gene Knockout: XDel technology maximizes on-target editing and minimizes off-target effects

Figure. A. XDel gRNAs achieve significantly higher on-target editing efficiency compared to individual sgRNAs across 7 genes in 6 cell types (including HEK293 and THP1). Data represent mean editing efficiency. Statistical significance determined by independent t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). B. XDel gRNAs exhibit significantly lower off-target editing across 63 predicted off-target sites in the same 6 cell types. Statistical significance assessed by t-test (*p < 0.05; **p < 0.01; ns = not significant).

Key takeaways

• EditCo Bio has developed a robust Cas9-mediated ex vivo gene editing method using unique XDel gRNA design technology that delivers guaranteed editing efficiency without pre-screening guide RNA activity.
• The consistent base pair distance spanned by XDel multiple gRNAs enables the use of targeted next-generation amplicon sequencing as a standard quality control method for genotyping Cas9-edited single-cell clones and cell pools.
• High-throughput automation allowed editing of 768 samples from multiple immortalized and primary cell lines followed by NGS analysis of 4,816 high-quality libraries to compare the on- and off-target editing efficiency of XDel’s multiple guide RNA editing strategy versus single-guide RNA editing.
• NGS results demonstrate that EditCo Bio’s XDel CRISPR editing strategy consistently achieves high on-target editing efficiency for gene knockouts compared to using a single-guide RNA while simultaneously minimizing editing efficiency at known off-target sites.
• This simultaneous high on-target editing with low off-target effects is consistent across various cell types, including immortalized cells, iPSCs, and primary cell types, enabling robust and reliable editing for a wide range of applications.