Gene Knockout Kits

Human and Mouse Knockouts Have Never Been Easier.

Common CRISPR knockout strategies rely on an individual guide RNA (gRNA) that produces random indels in the gene of interest. This approach produces a single double-strand break at the targeted locus and relies on the cellular repair mechanism to generate a frameshift mutation to disrupt the gene. This strategy is inefficient due to the wide variety and unpredictability of edits that can occur and often does not generate a functional knockout.

EditCo’s Gene Knockout Kit was designed to increase the efficacy of CRISPR-mediated knockouts. Typical guide RNA strategies rely on the generation of indels, which does not always result in complete gene disruption. EditCo’s multi-guide approach consistently generates fragment deletions at the targeted loci, so you can be confident in your gene knockout. 

Figure 1. EditCo’s Gene Knockout Kit was designed to increase the efficacy of CRISPR-mediated knockouts. 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 2. Multi-guide gRNA for each target resulted in >75% large deletion outcomes. Individual vs. multi-guide gRNA editing was compared for 2 gene targets (TNF, TLR4) in dendritic cells (transfected via nucleofection). Editing efficiency was analyzed by sequencing the targeted loci on a MiSeq and sequencing outcomes were categorized based on editing type (no indel, large deletion ≥50bp, small deletion <50bp, insertion). 

Consistent and Reliable Knockouts

Figure 3. Better knockout efficiency was found across 32 genetic targets assessed. Guide RNAs displayed 29.2% better median knockout efficiency when introduced in a multiguide format (89.9% KO score) relative to an individual sgRNA format (69.6% KO Score).

Fragment Deletions Result in Sustained Protein Depletion

Fragment deletions caused by multi-guide sgRNA editing result in protein knockout after seven days post-transfection in non-clonal cell lines.

Figure 4. Editing of three genes (CDK9, CINP, COASY) in HEK293 cells (transfected via nucleofection) using the multi-guide approach resulted in high knockout efficiencies, as indicated by Knockout (KO) Scores. Western blots showed loss of the corresponding proteins relative to mock treated cells (no sgRNA or Cas9). For seven days post-transfection, cells were assessed for the presence of the protein target via immunoblot analysis (GAPDH expression across the same timeframe was used for normalization).

Figure 5. 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.