This technical note summarizes guidance for designing CRISPR screens on the Cytos™ workflow (AVITI24™ system), covering the Optical Pooled Screening (OPS) and Multimodal Pooled Screening (MPS) configurations. It is a summary prepared for Velgen Technik; for the complete note, tables, and references, see the original Element Biosciences publication linked at the end. (Element Biosciences document LT-00084 Rev A, October 2026. For Research Use Only.)
Highlights
• Cytos chemistry on the AVITI24 reads sgRNA sequences directly from cells using Direct in Sample Sequencing (DISS), without poly-A capture.
• sgRNA identity can be linked to cell morphology, 3′ transcriptome data, and immunofluorescence protein detection at subcellular resolution.
• Good scaffold design, appropriate multiplicity of infection (MOI), and selection increase the CRISPR effect and the fraction of cells with a detected guide.
Introduction and Kit Configurations
Traditional pooled CRISPR phenotyping often captures only one modality and involves trade-offs between scale, coverage, and cost. Because sgRNAs are not naturally polyadenylated, transcriptomic methods need special capture and optical methods may need custom probes — challenges that DISS addresses by reading the guide directly.
• OPS (Optical Pooled Screening) Kit — DISS sgRNA reads with morphology metrics.
• MPS (Multimodal Pooled Screening) Kit — OPS plus 3′ transcriptome data.
• Cytos Protein Kit — immunofluorescence-style protein detection.
A QC panel targeting ACTB and GAPDH (4 guides each) is included with OPS and MPS kits as a guide-detection control.
Cytos CRISPR Screening Workflow
1. RT priming — a user-designed reverse transcription primer binds the 3′ flanking region of the target RNA.
2. cDNA synthesis — reverse transcription produces cDNA containing the target sequence.
3. Amplification — rolling circle amplification (RCA).
4. Sequencing — Avidite Base Chemistry with a user-designed sequencing primer.
5. Primer setup — the ElemBio Cloud Custom Designer automates primer design from user-specified scaffold sequences; primers are ordered from third-party oligo vendors and loaded into the 3′ Transcriptome Cartridge at run setup.
6. Cell assignment — a Target Cell Assignment (TCA) Manifest enables automated sequence-to-sgRNA and sgRNA-to-cell assignment. A Confidence Score indicates confident assignment to a single sgRNA target; by default a cell with multiple guides is assigned to a dominant spacer if more than 50% of its guides map to it (adjustable for systems with multiple guides per cell).
Scaffold Design and sgRNA Detection
The original sgRNA scaffold contains four consecutive uridines, which act as an RNA Pol III terminator and can cause premature transcript termination. Two modifications improve detection: changing the U-A pair at position 4 of the uridine run to a C-G pair, and extending the Cas9-binding hairpin by 5 base pairs.
Combining both changes raised sgRNA levels about 5–6 fold and gave the highest knockout efficiency. Element recommends using the optimized scaffold for Cytos runs; contact Element Biosciences Support for implementation help.
Benchmark: Scaffold and Vector Performance
Six libraries were benchmarked on a 12-well Cytos Slide Kit. Optimized-scaffold libraries yielded about 20–40 counts per cell, while original-scaffold libraries typically yielded fewer than 15. Guide expression and the fraction of unguided cells depend on MOI, passage number, cell type, and antibiotic selection.
| Library / Vector | Promoter | Scaffold | Cell Type | % Assigned | Mean counts/cell |
|---|---|---|---|---|---|
| CROP-seq-opti | U6 | Optimized | U2OS | 96.6% | 42.8 |
| PerturbView | U6 | Optimized | MCF7 | 70.6% | 22.1 |
| Cellecta pRSGUPS2 | U6 | Optimized | A549 | 86.4% | 20.7 |
| lentiGuide-Puro | U6 | Original | U2OS | 65.2% | 12.1 |
| pLentiCRISPRv2 | U6 | Original | A549 | 52.8% | 9.8 |
| pLentiCRISPRv2 | U6-T7 hybrid | Original | HeLa | 14.5% | 1.4 |
Practical Considerations
• Assignment planning — use a pilot experiment, or assume about 50% guide assignment as a starting point.
• Scale example — a 1-well flow cell recovering about 200,000 assigned cells could support up to 500 conditions at 400 cells per condition.
• Genome-scale example — a 2,000-gene screen at 400 cells per perturbation needs about 800,000 assigned cells; at roughly 50% assignment in a 1-well A549 flow cell (about 400,000 total cells), this is about 4 flow cells.
• These figures are illustrative — actual numbers depend on cell line, flow cell configuration, coverage variability, and the statistical criteria of the experiment.
• Example data — A549 cells with a 28-sgRNA library (6 genes plus a non-targeting control, 4 guides each) gave 109,972 assigned cells across 6 wells, with counts varying by target due to cloning efficiency, MOI, and phenotypic effects.
• Fluorescent reporters — plan reporter use for selection before the run, especially for protein or morphology readouts.
Summary
OPS and MPS workflows sequence sgRNA spacers directly from fixed cells using DISS, and guide identity can be combined with morphology, 3′ transcriptome, and immunofluorescence protein data at subcellular resolution. Key enablers include simplified primer design, automated analysis, and multimodal readouts for screens from focused to genome-wide scale.
Source
This is a summary of the Element Biosciences technical note “Cytos™ CRISPR Screening Guidance” (LT-00084 Rev A). All technical claims, data, and the benchmark table are from Element Biosciences. References include Dang, Y. et al., Genome Biol. 16, 280 (2015) and Chen, B. et al., Cell 155, 1479–1491 (2013). Read the full technical note at elementbiosciences.com. To discuss Cytos for your lab, view the Cytos product page or contact our team.