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CRISPR

Genome editing: precision starts with delivery

CRISPR endonuclease delivery has evolved through three formats in under a decade, and each change was driven less by editing biology than by delivery kinetics. How long Cas9 stays active in the cell determines both editing efficiency and off-target burden.

>90%
Indel rate achievable with RNP electroporation in activated T cells
~24 h
Time to maximal editing with RNP versus 48–72 h with plasmid
3 formats
Plasmid, mRNA + sgRNA, and pre-assembled RNP
01

Three delivery formats, three kinetics

Plasmid delivery is the legacy format: a single vector encoding Cas9 and the guide RNA, inexpensive and simple to produce. The problem is duration. Plasmid DNA persists for days, so Cas9 is expressed continuously and has far more opportunity to cut at near-match sites; selection pressure and plasmid integration add further risk in therapeutic settings.

mRNA delivery shortens the window to 12–24 h because the transcript is degraded once translated. Editing efficiency stays high and off-target activity drops, but mRNA is fragile, requires cold-chain handling during formulation, and the timing of sgRNA and Cas9-mRNA co-delivery has to be controlled — an excess of one component wastes the other.

RNP delivery — pre-assembled Cas9 protein with sgRNA — is the current standard for sensitive and therapeutically relevant cells. The complex is active within minutes of entering the cytoplasm, is degraded within hours, and never involves a nucleic acid encoding the nuclease, so there is no persistent expression and no integration risk.

02

Why immune cells changed the field

Primary T cells and haematopoietic stem cells resist lipid reagents, are exquisitely sensitive to innate immune activation, and divide slowly. Delivering plasmid DNA triggers cytosolic DNA sensing; delivering mRNA triggers RNA sensing unless the transcript is base-modified. RNP delivery avoids both sensors entirely, which is a large part of why it became the clinical default.

Electroporation with a cell-type-specific pulse program is the enabling technology. Nucleofection drives RNP through the nuclear envelope even in resting cells, and electroporation platforms now hold editing rates above 90% at the TRAC and PDCD1 loci in primary human T cells while preserving viability.

  • Pre-complex Cas9 protein with sgRNA at a 1:1 to 1:2 molar ratio and incubate 10 min at room temperature before electroporation.
  • Use chemically modified sgRNA with phosphorothioate ends to resist nuclease degradation in the cytosol.
  • Validate the guide in a reporter line or by T7E1/amplicon sequencing before committing precious primary cells.
  • Assess editing by amplicon deep sequencing (indel spectrum), not by a surrogate fluorescent readout alone.
03

Reagent and platform choices that work

For easy adherent lines where plasmid transfection is acceptable, lipid reagents formulated for CRISPR-sized cargo such as CRISPRMAX and Lipofectamine 3000 give high co-delivery of Cas9 and guide plasmids. For RNP in adherent cells, lipid reagents engineered for protein–RNA complexes perform well, and reverse transfection formats improve consistency.

For hard-to-transfect primary cells, nucleofection with RNP is the reference standard. Where scale matters — manufacturing-scale T cell engineering — flow electroporation in a closed assembly preserves the electric dose as cell numbers rise into the billions.

Workflow

How a working experiment is built

  1. 1

    Design and validate the guide

    Select guides with on-target scores above 0.6, screen for off-target near-matches, and validate cutting in a surrogate line before touching the target cells.

  2. 2

    Assemble the RNP

    Mix Cas9 protein with modified sgRNA and incubate 10 min at room temperature. Keep on ice until delivery and use within the working day.

  3. 3

    Deliver by the format the cell demands

    Lipid reagent for adherent lines, nucleofection for primary and non-dividing cells, flow electroporation for manufacturing scale.

  4. 4

    Recover properly

    Warm, cytokine- or serum-containing recovery medium added immediately after the pulse is the single biggest determinant of viability in immune cells.

  5. 5

    Quantify the edit, not the reporter

    Amplicon sequencing at 48–96 h gives the indel spectrum; a fluorescent surrogate alone cannot distinguish a clean edit from a large deletion.

Failure patterns

What usually goes wrong, and the fix

High cutting in a reporter cell line, poor editing in primary cells

Fix: Switch format — RNP electroporation rather than lipid-delivered plasmid; validate the program for the primary cell type

Editing efficiency falls as cell number increases

Fix: Keep the electric dose constant and check that RNP per cell, not per tube, is held constant

Large deletions dominate the indel spectrum

Fix: Shorten Cas9 exposure — move from plasmid to RNP — and confirm with long-range PCR

Sudden drop in viability 24 h after nucleofection

Fix: Increase recovery time in warm medium, reduce cell density during recovery, and confirm the buffer is not expired

The other two applications

Provenance

References for this application

Every protocol on this page was reconstructed from the manufacturer’s own documentation. Use these links to confirm concentrations, catalogue numbers and cell-line-specific variants before running the experiment.

  1. [1]Thermo Fisher — CRISPR-Cas9 genome editing delivery options
  2. [2]Integrated DNA Technologies — Alt-R CRISPR-Cas9 RNP electroporation protocols
  3. [3]Lonza — Nucleofection protocols for CRISPR RNP delivery to primary cells
  4. [4]MaxCyte — ExPERT flow electroporation for cell therapy engineering

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