Key Takeaways
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XDel maintained robust protein depletion across four membrane targets in K562 cells, with depletion sustained for up to 3 weeks post-transfection.
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NGS genotyping revealed stable large-fragment deletion profiles across multiple passages, with robust protein depletion maintained despite a high proportion of in-frame deletions.
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Compared with single-guide and two-guide CRISPR approaches, XDel achieved greater knockout performance, including 95% CD46 protein depletion versus 70% for a two-guide competitor combination (p < 0.05), while generating larger targeted deletions.
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XDel simplifies genotyping by requiring only one primer pair for Sanger or NGS sequencing, reducing time, cost, and workflow complexity compared with conventional two-guide approaches.
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Single-vial delivery streamlines workflow by eliminating guide resuspension, normalization, and guide-mixing steps required for conventional multi-guide designs.
Figure. XDel enables sustained protein depletion with a simplified genotyping workflow. High-level comparison of the advantages of the XDel approach versus conventional single-guide and two-guide CRISPR knockouts. *Note: Adding a third guide would increase workflow complexity further by introducing another vial that must be combined and normalized and would require a third primer pair and sequencing reaction.
Background
Many commercial CRISPR-based editing systems use one to two guide RNAs to disrupt protein expression by generating insertions and deletions (indels) that shift the protein reading frame. However, DNA repair mechanisms can sometimes generate in-frame deletions — whose lengths are multiples of three nucleotides — that remove amino acids without disrupting the reading frame, potentially leaving the protein partially or fully functional.
When gene editing fails to completely eliminate target protein expression, confidence in the interpretation of downstream phenotypes diminishes.
EditCo's XDel technology is designed to address this by generating larger deletions that result in high, sustained protein depletion. XDel uses up to three modified sgRNAs positioned close together within a single early exon, working cooperatively to create large, targeted fragment deletions of 21+ bp.
Figure. XDel’s unique editing strategy generates large deletions, resulting in high editing efficiency and sustained protein depletion. XDel uses up to three modified sgRNAs (light gray) targeting a single early exon in the gene of interest. When co-transfected, these sgRNAs create concurrent double-stranded breaks (vertical dotted lines) at the targeted genomic locus and consequently induce one or more 21+ bp fragment deletions.
Experimental design
Four surface membrane proteins — CD46, CD55, CD59, and CD82 — were knocked out in human immortalized K562 cells using XDel designs. Transfection was performed in duplicate (n = 2), with each biological replicate split into 3 technical replicates post-transfection (n = 6).
Figure. Time-course analysis through flow cytometry demonstrates a high level of membrane protein knockout that remains consistent over time. (A) Four membrane proteins were targeted for knock-out in K562 cells using XDel technology, following EditCo Bio’s proprietary workflow. Transfection was performed in duplicate (n = 2), and each biological replicate was subsequently split into 3 technical replicates post-transfection (n = 6). Live cells were collected for flow cytometry analysis on days 3, 7, 14, and 21 post-electroporation. Antibodies against CD46, CD55, CD59, and CD82 were used to detect the expression of these surface membrane proteins on K562 cells at 4 specified time points on edited, Cas9-only, and non-transfected samples. Cell viability was measured using viability dye DRAQ7. Figure created with BioRender. (B) Bar plots showing high protein depletion and viability across two biological replicates for each of the four chosen gene targets. Tight error bars indicate high reproducibility of the XDel approach. (C) Representative flow cytometry histograms of CD55 and CD59 protein expression.
- Protein expression measured by flow cytometry at days 3, 7, 14, and 21 post-electroporation
- Editing efficiency quantified by NGS using EditCo's Eos NGS Library Preparation workcell and proprietary NGS analysis tool
- Cell viability measured using viability dye DRAQ7
Results
Sustained protein depletion across four targets
Flow cytometry revealed greater than 90% protein depletion across all four membrane targets at all time points, persisting for up to 3 weeks post-transfection. Cell viability remained high throughout. Tight error bars across replicates confirm high reproducibility of the XDel approach.
Stable indel profiles by NGS
NGS genotyping of CD55 and CD59 knockouts showed observed indel lengths of ≥54 bp — consistent with large fragment deletions expected from XDel's multi-guide strategy. The proportion of individual indels remained stable across multiple cell passages. Notably, despite a high proportion of in-frame deletions, complete loss of detectable protein expression was observed and persisted over time — suggesting large in-frame fragment deletions through XDel's multi-guide strategy are sufficient to cause sustained protein depletion.
XDel vs. single-guide: higher efficiency, larger deletions
Editing efficiency of CD46-targeted XDel guides was compared with individual Vendor X sgRNAs:
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XDel: complete loss of detectable CD46 protein, fragment deletions ≥36 bp
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Vendor X sgRNA 1 and 2: only partial CD46 protein loss, low editing efficiency, indels ≤18 bp
XDel generated a higher proportion of in-frame deletions than the single guides, yet still achieved complete protein loss — confirming that deletion size is a critical determinant of functional knockout outcomes.
XDel vs. two-guide: 95% vs. 70% protein depletion
The two-guide combination's distant guide placement requires two primer pairs and separate sequencing reactions — and creates a potential blind spot for fragment deletions outside the sequencing window of either primer pair. XDel's compact guide positioning eliminates this risk.
