title: "Literature review: Chen et al. 2025 — multiplex CRISPR screening in primary T cells" project: research-lab-template tags:

• literature
• crispr
• immunology date: 2026-04-07

Chen et al. 2025 — multiplex CRISPR screening in primary T cells

Citation (hypothetical for template): Chen, L. et al. Nat. Methods (2025). DOI: 10.xxxx/nmeth.2025.xxx

One-line summary

The authors combine pooled CRISPR perturbations with single-cell RNA-seq in ex vivo human T cells, reporting improved guide detection sensitivity versus prior plate-based assays.

Key findings

• Multiplexing ~120 guides per donor while retaining >70% cell viability at 72h under their electroporation conditions.
• A computational demultiplexing step reduces doublet-induced false positives; code is available (check license before reuse).
• Validation with orthogonal flow cytometry on top hits matches directionality in four of five targets tested.

Methods worth noting

• Electroporation parameters and media supplements are specified in supplementary tables—useful if we replicate stress controls.
• They normalize by non-targeting guides using a trimmed mean; may bias rare phenotypes.

Relevance to our lab

Directly applicable if we pivot the cytokine secretion screen to primary cells. Our current immortalized line protocol will not transfer without revisiting viability curves and electroporation load.

Open questions

• Donor batch effects: only six donors in main figures.
• Long-term culture effects on editing outcomes not addressed beyond 96h.

Next step: Pilot three guides from their positive control set in our hands before scaling the library.