CRISPR-edited Immortalized Knockout Cells

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.

EditCo’s Automated Immortalized Cell Line Editing Process

Our gene editing workflow includes the use of our unique multi-guide design 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.

Our Unique Guide Design Powers Functional Knockouts and Sustained Protein Depletion

Figure 1. 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.

Hundreds of Diverse and Efficient Knockouts Generated in Under 4 Weeks

Figure 2. Express Cell Pools generated across 355 genes and 36 different cell lines in just 12 days. Using EditCo’s multi-guide designs, up to 3 modified gRNAs per target were conjugated with SpCas9, and RNPs were transfected into cells. On average, Express Knockout Cell Pools were generated in 12 days and achieved >80 percent Gene Knockout (Knockout Score percentage).

Sustained Gene Knockout Across Cell Passages

Figure 3. 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

Figure 4. 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.

Figure 5. 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.

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 (green 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 fragement 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.