CRISPR-edited Immortalized Knockout Cells

EditCo’s Automated Immortalized Cell Line Editing Process

200 Ways We Optimize CRISPR to Achieve High Editing Across Cell Lines

EditCo’s 200-point optimization has been performed in over 100 human cell lines across 15 tissue types to identify the best parameters that yield the highest editing efficiency for each cell line.

Our gene editing workflow includes the use of our unique XDel Technology or single-guide transfection, genotyping, and sequencing analysis. Choose from either EditCo supplied cell lines banked from our high-quality cell supplier ATCC, or onboard your cells.

XDel delivers higher and more consistent on-target editing compared to single-guide, ensuring superior editing performance

While traditional single-guide knockouts demonstrate a wide and often unpredictable range of on-target editing efficiencies, EditCo XDel technology stands out for its consistent high performance. XDel ensures reliable outcomes across many different cell lines, often outperforming single-guide in both efficiency and reproducibility.

Figure 1.  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.

XDel technology maximizes on-target gene editing while minimizing off-target effects compared to single-guide editing

Unlike single-guide RNA strategies, which show variable editing efficiencies across different doses, EditCo’s XDel technology consistently achieves high on-target editing efficiency - even at low does concentrations. This reproducibility highlights the robustness of XDel technology which delivers reliable results independent of dose or position effects as seen by the single-guide approach. XDel gRNA design ensures superior and reproducible editing performance, allow you to focus on your research without the need for additional optimization.

Figure 2.  XDel multiple guide RNA 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 3.  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 off-target 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.

XDel Cell Pools Demonstrate Sustained Gene Knockout Across Multiple Cell Passages

Figure 4. High editing efficiency is sustained through multiple cell passages. Using EditCo’s multi-guide designs, up to 3 modified gRNA per target (across 6 genes) were conjugated with SpCas9, and RNPs were transfected into U2OS cells. Editing efficiencies were measured at different time points post-transfection and up to 4 passages. The median editing efficiency is >85 percent for each of the targets. 

Cell Viability Is Maintained Across Passages with XDel

Figure 5. Cell viability is maintained through multiple cell passages in U2OS cells. Viability was taken using Celigo. Please note that CDK9 is a common essential gene, hence the lower viability and cell number observed along the passages.

XDel knockout cell pools show persistent protein depletion, enabling  their direct and reliable use in functional assays

Figure 6. Edited cell pools utilizing the multi-guide technology shows persistent protein depletion. This enables direct and reliable use of these cell pools for functional assays. 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 multi-guide sgRNA and Cas9 (as RNPs). A negative control pool was also transfected for each target using non-targeting sgRNA.

How does the XDel strategy work?

Traditional CRISPR knockout methods often depend on a single guide RNA that, in tandem with SpCas9, generates assorted insertions or deletions (indels) at the target cut site. This approach can be unpredictable, leading to variable edits and incomplete knockouts.

EditCo’s smart informatics generates a multi-guide design which is composed of up to 3 sgRNAs targeting a single gene of interest. The guides are spatially coordinated and work cooperative to induce a guided repair that results in fragment deletion in an early exon, making it the most reliable knockout strategy compared to other pooled strategies. 

Figure 7. XDel design includes up to 3 modified sgRNAs (purple bars) that target a single gene of interest. When co-transfected, the sgRNAs create concurrent double-stranded breaks at the targeted genomic locus and consequently induce one or more 21+ bp fragment deletions. 

Efficient Generation of Knockout Clones with Functional Outcomes

EditCo’s CRISPR Knockout Cell Clones eliminate the time and hassle of doing CRISPR editing and creating clonal cell lines so you can focus on your research.

Validate your Assays with up to 95% Protein Knockout

EditCo’s Knockout Cell Pool gives you the protein knockout you need in the cell line you want, enabling you to either assay pools directly or quickly move to isolate single cell clones.

Figure 6. EditCo’s Knockout Cell Pool achieved 95 percent protein knockout without clonal expansion. A cell pool with a surface protein knocked out was generated in U2OS cells with a Knockout Score of 87 percent (left panel, which is the percentage of indels that either introduce a frameshift mutation or are larger than 21 nucleotides, analyzed by ICE). Levels of this protein were analyzed by flow cytometry (blue) and were reduced by 95 percent relative to wild type (green, right panel). Negative control samples were stained with an isotype control antibody (purple). Data provided by Eurofins-DiscoverX.

Engineered Cell Libraries Demonstrate High Editing Efficiencies Across Cell Types

Engineered Cell Libraries provide a set of CRISPR-edited cell pools that can be used directly in functional assays for a wide array of different screening and discovery applications. Editing efficiencies for each pool are assessed by sequencing and ICE analysis to inform the interpretation of assay results.

Knockouts in Human Immortalized Cell Line

Figure 1. A library of knockout cell pools corresponding to 42 gene targets was generated via high-throughput editing in human cells. Data from the experiment are shown above. An Engineered Cell Library with 42 gene targets was generated via high-throughput editing of an immortalized-adherent human cell line, A375, using multi-guide technology. The data demonstrate both indel frequencies and Knockout Scores. Of the 84 percent of pools successfully sequenced and analyzed, an average indel frequency of 99 percent and an average Knockout Score of 98 percent were observed.

Knockouts in Mouse Immortalized Cell Line

Figure 2. A library of knockout cell pools corresponding to 192 gene targets was generated via high-throughput editing in mouse cells. The data demonstrate both indel frequencies and Knockout Scores. Among the 80 percent of pools successfully analyzed, the average indel frequency was 89 percent, while the average Knockout Score was 84 percent.

Our Unique Guide Design Powers Functional Knockouts and Sustained Protein Depletion

The multi-guide approach consists of up to 3 gRNAs (purple bars) spatially coordinated that target a single early exon of a gene and induce multiple concurrent double-strand breaks in the target sequence. This results in disruptive one or more >21 bp fragment deletions. This approach is associated with higher frequencies of knockout alleles in edited pools but reduced occurrence of off-target edits as compared to single-guide approaches.

Figure 3. The multi-guide approach consists of 3 gRNAs spatially coordinated to create a fragment deletion in a single exon. Targeting a single early exon of a gene induces multiple concurrent double-strand breaks in the target sequence of a gene. This results in disruptive one or more >21 bp fragment deletions. This approach is associated with higher frequencies of knockout alleles in edited pools but reduced occurrence of off-target edits as compared to single-guide approaches.

Figure 4. Edited cell pools utilizing the multi-guide technology shows persistent protein depletion. This enables direct and reliable use of these cell pools for functional assays. 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 multi-guide gRNA and Cas9 (as RNPs). A negative control pool was also transfected for each target using non-targeting gRNA.