Abstract
Adoptive cell transfer (ACT) is an important approach for basic research and emerges as an effective treatment for various diseases including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. Here, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms (SNP) in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology directed repair (HDR), systematically optimize experimental parameters and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Both repairing the cause of the scurfy syndrome, a 2bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.
Footnotes
1 This work was supported by grants of the Swiss National Science Foundation (SNSF Professorship PP00P3_144860 to LTJ) and the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health, USA, under Award Number R56/R01AI106923 (to LTJ) and a fellowship from the Fonds de Recherche Santé Québec, Canada (to MK). The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
2 M.K. performed and analyzed all experiments in the manuscript. R.M. performed cloning of PCR products for sequencing and analyzed sequencing data, helped cloning and depositing plasmids, helped to write materials and methods and to prepare the supplementary tables; M.K. and L.T.J. designed the experiments, interpreted the data, discussed results and wrote the manuscript.
3 Data and materials availability: All plasmids used in the manuscript are deposited at addgene.org
4 Address correspondence and reprint requests to Lukas T. Jeker, MD PhD, Assistant Professor of Experimental Transplantation Immunology & Nephrology, Molecular Immune Regulation, Lab 313, Department of Biomedicine, Basel University Hospital and University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland; e-mail: lukas.jeker@unibas.ch
5 The online version of this article contains supplemental material.
6 Competing interests: M.K. and L.T.J. have filed provisional patent applications related to this work.
7 Abbreviations used in this article: ASA, allele switching assay; ACT, adoptive cell transfer; CAR, chimeric antigen receptor; Cas9, CRISPR associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; crRNA, CRISPR RNA; DSB, double strand break; dsDNA, double stranded DNA, gRNA, guide RNA; HDR, homology directed repair; hHSC, human hematopoietic stem cells; IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked; LCMV, lymphocytic choriomeningitis virus; NHEJ, non homologous end joining; PAM, protospacer adjacent motif; RNP, ribonucleoprotein; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; ssDNA, single stranded DNA; tracrRNA, trans-activating crRNA;