Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells.

Oh SA, Seki A, Rutz S. 
Source: Curr Protoc Immunol
Publication Date: (2019)
Issue: 124(1): e69
Research Area:
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
T cell, mouse - BALB/c
Species: mouse
Tissue Origin: blood
T cell, mouse - C57BL/6
Species: mouse
Tissue Origin: blood
T cell, mouse, stim
Species: mouse
Tissue Origin: blood
4D-Nucleofector™ X-Unit

Wash T cells (from Basic Protocol 1 or Basic Protocol 2) in PBS to remove tracesof FBS, which can contain RNAses.
3. Suspend T cells at 2 × 106 cells per 20 µl nucleofection buffer (included in the
Primary Cell 4D-Nucleofector X Kits).
A wide range of cell numbers (between 105 to 107 cells per nucleofection) can be used
without loss in knockout efficiency or viability.
For human T cells: P2 nucleofection buffer.
For mouse T cells: P4 nucleofection buffer.
T cells should be suspended in nucleofection buffer immediately before nucleofection, as
the nucleofection buffer is toxic to cells.
Work carefully but as quickly as possible from steps 3 to 11 to minimize the amount of
time the T cells are in the nucleofection buffer.
4. Add 5 µl of RNP (obtained following Basic Protocol 3), and mix by repeated
If using a pool of RNPs (we have tested up to 6 RNPs), reduce the volume of each RNP
to 3 µl. The entire volume of the cell/RNP mix is then used in subsequent steps.
5. Incubate 2 min at room temperature.
6. During the incubation, turn on the 4D-Nucleofector machine.
7. Choose the correct preset program for electroporation.
a. For human T cells: Primary Cell P2.
b. For mouse T cells: Primary Cell P4
8. Enter the appropriate pulse code.
a. For human T cells: EH100.
b. For mouse T cells: DS137 for resting cells; and CM137 for activated cells.
9. Transfer the T cell/RNP mix to Nucleocuvette strips.
Pipet gently to avoid air bubbles.
10. Place cuvette strips in the 4D-Nucleofector machine, and electroporate using the
program and pulse code selected in steps 7 and 8.
11. Remove cuvette strips from the 4D-Nucleofector machine and the 96-well plate with
pre-warmed medium from the 37°C incubator. Immediately add 100 µl pre-warmed
T cell medium to a nucleofection cell, mix gently by pipetting once or twice, and
transfer the entire volume (25 µl T cells and RNPs plus 100 µl T cell medium) from
the nucleofection cell to the remaining 100 µl of T cell medium in the appropriate
well of the 96-well plate.
When using more than 2 × 106 cells per transfection, T cells should be cultured in
multiple wells at a max. of 2 × 106 cells per well.
12. Incubate the 96-well plate containing the electroporated cells at 37°C incubator.
Allow electroporated T cells to recover for 2 hr before any further manipulation.

Lonza summary:

Detailed method paper using Nucleofector 4D  to transfect CRISPR Cas9 RNPs into non stimulated (and stimulated) mouse and human T cells.

Showing optimization of RNPs quantity in a Nucleofection experiment and the use of an enhancer (from IDT) to reduce RNPs amounts. The authors identified P2 / EH-100 as optimal Nucleofection conditions for human T cells (stimulated and non-stimulated), P4 / DS-137 for the mouse unstimulated T cells and P4 / CM-137 for the mouse stimulated T cells.

Earlier work from the same last author presented this approach in a research paper (Seki, et al. J Exp Med. 2018)


CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleoprotein (RNP) transfection of primary mouse and human T cells without the need for TCR stimulation that achieves near complete loss of target gene expression at the population level. This approach enables rapid target discovery and validation in both mouse and human primary T cells.