With years of experience and hundreds of thousands of successful edits, this journey has given us invaluable insights into one of the biggest challenges scientists face in the CRISPR knockout space: the extensive optimization and rework often required to confidently achieve a gene knockout.
Recognizing this challenge, we asked ourselves: How can we use our expertise to design a more efficient and reliable method to knock out genes? By focusing on this mission, EditCo has developed innovative solutions that streamline the knockout process, empowering researchers to achieve their goals with greater confidence.
EditCo’s Solution for Better Knockouts: XDel Technology
Figure 1. Multiple guides would cooperatively together to create a large deletion in a single gene exon. EditCo’s XDel guide design includes up to 3 modified sgRNAs (grey bars) that target a single gene of interest. When co-transfected, the sgRNAs create concurrent double-stranded breaks (vertical dotted lines) at the targeted genomic locus and consequently induce one or more 21+ bp fragment deletions.
To address the inefficiencies of traditional CRISPR knockout strategies, we developed a solution: a set of predesigned guide RNAs (gRNAs) targeting a single exon of a gene that work cooperatively to increase editing efficiency by creating targeted fragment deletions. Unlike the standard approach, which relies on a single gRNA to introduce random indels at one site—or multiple gRNAs that randomly target different regions—our method is specifically designed to create coordinated deletions that reliably disrupt the gene. This approach improves the likelihood of generating a complete functional knockout, reducing the unpredictability and inefficiency associated with traditional strategies.
By analyzing 7 endogenous loci in 4 cell lines, XDel knockout technology delivers significantly higher and more consistent on-target editing compared to single-guide, ensuring superior editing performance in immortalized cells and iPSCs.
Figure 2. XDel multiple gRNA on-target editing efficiency is significantly higher and more consistent compared to single-guide RNA methods. On-target editing efficiency of multi-guide (pink bars) vs their 3 respective single-guide RNAs (blue bars) across 7 endogenous target loci transfected in two immortalized (IMM) and two iPSC cell lines indicates that XDel technology delivers significantly higher and more consistent on-target editing efficiency compared to single-guide. Each transfection was performed in triplicate and data were analyzed via NGS by EditCo’s proprietary software analysis.
Derisking Unintentional Edits: XDel Knockout Cells Actually Have Fewer Off-target Edits Than Single-guide
A common concern with multi-guide approaches is the potential for off-target edits. To address this, we rigorously tested XDel Knockout Cells to ensure it produces high-quality knockouts with minimal off-target effects. Our data shows that XDel guide designs not only maximize on-target gene editing efficiency but also significantly minimize off-target edits compared to single-guide strategies.
Figure 3. XDel multiple gRNA off-target editing efficiency is significantly lower compared to single guide RNA methods. Off-target editing efficiency of XDel cells (pink bars) at all 3 of their respective single-guide off-target sites vs their 3 respective single-guide RNAs (blue bars) across 63 off-target loci (3 off-target loci per 7 on-target site tested) transfected in two immortalized (IMM) and two iPSC cell lines indicates that XDel cells deliver significantly lower off-target editing efficiency compared to single-guide. Each transfection was performed in triplicate and data were analyzed via NGS by EditCo's proprietary software analysis.
Figure 4. XDel multiple gRNA off-target editing efficiency is minimal compared to single guide RNA methods. Off-target editing efficiency of multi-guide (pink dots) vs their respective single-guide RNA (blue dots) at 46 off-target loci observed to have positive off-target editing in any of the four tested cell lines indicates that multi-guide delivers significantly lower off-target editing efficiency compared to single-guide. Each off-target site is in triplicate, 193 total data points analyzed via NGS by EditCo’s proprietary software analysis.
XDel Knockout Cells show persistent protein depletion, enabling their direct and reliable use in functional assays
Achieving a functional knockout is essential to be able to use edited cells for downstream assays. Edited cell pools generated using our XDel gRNA design demonstrate persistent protein depletion, a key indicator of effective gene disruption. This ensures that the cell pools can be directly used in functional assays without requiring additional validation or rework and provide reliable long-term functional data.
Figure 5. Protein depletion remains consistent across a variety of gene knockout cell lines. Western blot analyses for all 5 knockout target genes indicate a complete depletion of proteins across the 3 specified time points (days 7, 14, and 21) relative to the negative controls. Knockout cell pools for the target genes were generated by nucleofecting U2OS cells with sgRNA and Cas9 (as RNPs). A negative control pool was also transfected for each target using non-targeting sgRNA.
Off-target editing is minimized, in part, due to lower gRNA concentration made possible through synergistic multi-guide cooperation
The cooperative effect of XDel guide design allows for lower gRNA concentration while maintaining high editing efficiency and minimizing unwanted off-target editing.
Figure 6. XDel multiple guide RNA editing efficiency remains high as gRNA concentration is decreased compared to single guide RNA methods. Average on-target (pink bars, multi-guide; blue bars, single-guide) editing efficiency in transfected HEK293 cells with multi-guide (left) vs single-guide (right) and SpCas9 at increasing RNP concentrations (0.25X, 1X, 4X) at 7 endogenous sites vs off-target editing efficiency (stacked grey bars) at 63 offtarget sites indicates that XDel cells maintain high on-target editing efficiency with minimal off-target effects compared to single-guide. Each transfection was performed in triplicate and data were analyzed via NGS by EditCo's proprietary software analysis.
Doing a CRISPR screen and want to skip ahead?
Our high-efficiency predesigned guides, combined with optimized CRISPR workflows and automation, create the ideal combination for producing high-quality, rapid and reliable knockout edits. This seamless integration ensures that researchers can achieve consistent results at scale, reducing variability and accelerating their research.
Figure 7. EditCo’s automated pipeline enables XDel Knockout Cell pools in high-throughput library format. Ideal for loss-of-funtion screening without requiring extentive editing work to build your arrayed knockout cell library.
Learn More and Take the Next Step
Whether conducting gene function studies, modeling diseases, or drug discovery, EditCo is your trusted partner in CRISPR knockout research. Our innovative approach delivers:
- High Efficiency: Achieve higher on-target editing rates and consistent knockout performance
- Reliable Results: Persistent protein depletion, validated through function assays
- Enhanced Reproducibility: Minimize variability for dependable outcomes across experiments
Ready to accelerate your research with robust gene knockouts?
- Learn more about XDel Knockout Design in Immortalized Cells and iPS Cells
- Contact Us
