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

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Figure: XDel gRNA results in significantly high consistent on-target editing efficiency compared to single-guide RNAs across multiple cell types. Bar chart of average on-target editing efficiency (y-axis) of XDel guide design (pink) vs individual sgRNA (dark blue) across 7 genes in 6 cell types (Immortalized includes HEK293 and THP1 cells, x-axis). Stars indicate statistical significance, independent t test (* = p<0.05, ** = p<0.01,*** = p<0.001, ****=p<0.0001).

Key takeaways:

• EditCo Bio has developed a robust Cas9-mediated ex vivo gene editing method using unique XDel gRNA design technology that delivers guaranteed high knock-out levels 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.