Hard-to-Transfect Cells: A Delivery Strategy Guide

Four properties, and they usually come together:

A primary T cell has three of these; an iPSC has one; a neuron has two. The strategy follows from which ones apply.

The pattern: electroporation of RNP dominates for cells you can dissociate, and viral delivery dominates where you cannot.

1. RNP electroporation, with a dose and pulse optimisation on a control guide.
2. mRNA, if RNP toxicity is limiting.
3. Lentivirus, if stable expression is the goal rather than a single edit.
4. AAV, for post-mitotic cells or in vivo work.
5. Editing an easier intermediate — progenitors, or a parental line — then differentiating.

Step 5 is the most under-used. For neurons, editing iPSCs and then differentiating is usually faster and cleaner than editing mature neurons.

• • Nucleofection buffer and pulse, more than payload dose.
• • Cell density and recovery medium immediately after electroporation.
• • Passage and confluency at the time of editing — a week of difference in culture age can double efficiency.
• • Guide quality: a validated guide makes a mediocre protocol look good, and vice versa.

• • A specific efficiency number before a pilot in your cells.
• • Clonal lines from primary cells — usually not feasible; we plan pool-based work.
• • Knock-in from HDR in post-mitotic cells at useful rates.
• • Editing organoids without transient structural disruption.

Stating these early is cheaper than discovering them at week six.

Two weeks, one control guide and one target guide, four conditions (two doses × two pulse settings), read out by amplicon sequencing and viability. The output is a working protocol, an efficiency range, and a decision on whether clonal work is realistic in that cell type.

Tell us the cell type, the edit, and what you have already tried. We will return a delivery plan with the pilot design and the expected decision point.

Contact EDITGENE to request a project consultation or quote.

Efficiency varies by cell source, passage, and protocol; nothing here substitutes for a pilot in the specific cells.

Delivery optimisation changes viability as well as editing rate; both should be reported.

• • Kim S, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research. 2014;24:1012–1019.
• • Shifrut E, et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell. 2018;175:1951–1963.
• • Drost J, Clevers H. Organoids in cancer research. Nature Reviews Cancer. 2018;18:407–418.