Arrayed CRISPR gRNA Libraries

Discover the New Way to Screen with XDel Technology

Other loss-of-function screening libraries require labor-intensive preparation, complex data analysis, and suffer from poor editing efficiency. Low-quality reagents can also be cytotoxic to sensitive cell types like primary cells. EditCo’s Arrayed CRISPR Synthetic Guide RNA Libraries are different.

Our XDel technology was designed to address the inefficiencies of traditional CRISPR knockout strategies. A set of predesigned guide RNAs (gRNAs) target a single early exon of a gene and work cooperatively to increase editing efficiency by creating targeted fragment deletions. Unlike standard methods that introduce random indels, our approach ensures coordinated deletions for more reliable gene disruption and complete functional knockouts.

Our libraries are ready-to-transfect, either complexed into RNPs or directly into a Cas9-expressing cell line avoiding long preparation protocols. The arrayed format simplifies screening by removing the need for NGS deconvolution, enabling binary and multiparametric functional assays.

Additionally, the XDel design allows the knockout gene fragments to be rapidly quantified using standard PCR amplification for seamless integration into your CRISPR workflow. Analyze your edits in seconds with Sanger sequencing using our ICE tool.

Figure 1. EditCo Arrayed gRNA Library Workflow. Incorporating smart guide design with automation coupled with our high-quality control, we plate only the best-quality sgRNA into each well with high precision enabling you to reliably knock out your targets in your screens with confidence, we guarantee it (excluding non-essential genes). Due to our optimized workflow, we can deliver your Arrayed CRISPR gRNA Libraries in as few as 7 days at unrivaled speed.

What We Provide

Available Gene Pathways

What We Provide

What We Provide

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 2. XDel 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 at the targeted genomic locus and consequently induce one or more 21+ bp fragment deletions.

XDel Technology Consistently Induces Fragment Deletions

Figure 3. XDel multiple gRNA creates fragment deletions. Individual vs. XDel 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). Data generated by Dr. Marco Jost and Dr. Jonathan Weissman, University of California, San Francisco, and Dr. Amy Jacobson and Dr. Michael Fischbach, Stanford University.

XDel Achieves Higher and More Consistent On-Target Editing in Immortalized, iPSC, and Primary Cells Compared to Single-Guide

Figure 4. 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 (with known high off-target editing sites) 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

Figure 5. XDel multiple guide RNA off-target editing efficiency is significantly lower compared to single guide RNA methods when targeting endogenous sites known for their high off-target levels. 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 sites 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 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 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.

Edited Cells with XDel Technology Demonstrate Sustained Gene Knockout Across Multiple Cell Passages

Figure 7. 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 8. 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 Technology Result in Sustained Protein Depletion

Figure 9. Protein depletion remains consistent across a variety of gene knockout cell lines. 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.

Achieve Robust Genotype and Phenotype Screening Results with XDel

Figure 10. Across 77 genes, arrayed XDel library resulted in an average Knockout Score of 75%. Using our multi-guide algorithm, up to 3 modified gRNA per target (across 86 genes) were transfected into U2OS-Cas9 expressing cell line using nucleofection. This resulted in an average Knockout Score of 75% for 77 of the genes. Nine of the 86 genes failed due to sequencing errors.

EditCo Libraries Enables High Editing Efficiencies in a Large-scale Primary Cell Screen

Figure 11. 97% median editing efficiency using across 280 loci in primary dendritic cells. XDel gRNA was transfected into primary dendritic cells using nucleofection. Editing efficiency was assessed via NGS across 280 loci. Data courtesy of Weissman Lab, UCSF.