Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

Authors:
Foss DV, Muldoon JJ, Nguyen DN, Carr D, Sahu SU, Hunsinger JM, Wyman SK, Krishnappa N, Mendonsa R, Schanzer EV, Shy BR, Vykunta VS, Allain V, Li Z, Marson A, Eyquem J, Wilson RC
In:
Source: Nat Biomed Eng
Publication Date: (2023)
Issue: 5: 647-660
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Basic Research
Molecular Biology
Cells used in publication:
B cell, human
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Natural killer Cells (NK), human
Species: human
Tissue Origin: blood
Culture Media:
Platform:
4D-Nucleofector® X-Unit
Experiment

RNP electroporation (T, B and NK cells)
In a 4D nucleofector (Lonza), 50 pmol of Cas9, Cas12a or ABE RNP was electroporated into 200 × 103 T, B or NK cells resuspended in 20 µl of P3 buffer and supplement (Lonza V4XP-3032) using the EH-115 pulse code. Cells were incubated for 10 min at 37 °C, then rescued with 80 µl of pre-warmed culture medium before diluting for further cell culture as above. For AAV knock-in experiments, 60 pmol of RNP was electroporated into 0.5 × 106 or 1 × 106 T cells per well.

Abstract

CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor a constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.