Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9+ T Cells.

Snigdha Majumder,Andreas Rosenwald,Luisa Bell,Friederike Berberich-Siebelt,Nadine Hundhausen,Dimitrios Mougiakakos,Andreas Beilhack,Rishav Seal,Domenica Saul,Isabelle Jugovic

doi:10.3389/fimmu.2021.683631

Abstract

Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3+ T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9+CD3+ T cells, CD4+ and CD8+ conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9+CD3+ T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.

Highlights

Until today, immunological studies depend on mouse models, which provide a manipulable systemic approach
To investigate whether gRNA-only is as efficient as RNPmediated knockout in naive mouse T cells collected from L2G85.CD90.1 mice, we prepared RNPs using 10 μg recombinant Cas9 protein and 150 pmol gRNA [3]
We fixed our protocol for 150 pmol gRNA and RPMI, but verified this in an extended approach comparing RNP and gRNA-only nucleofection of unstimulated and by anti-CD3, anti-CD28 and IL-2 prestimulated CD3+ T cells from wild type (WT) and Cas9+ mice for 72 hours

Summary

Introduction

Immunological studies depend on mouse models, which provide a manipulable systemic approach. In order to understand cause and consequence of gene function in health and disease, multiple transgenic mice have been created. This is tedious, modern Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 nuclease (CRISPR/ Cas9)-mediated techniques have improved the procedure enormously [1, 2]. Some insights can be gained if transgenic cells are analyzed in vitro or transferred to new mice. Models of T-cell gRNA Nucleofection of Naive Cas9+CD3+ T Cells transfer serve translational purposes. Any manipulation of T cells in GvHD models represents an idea to avoid or limit GvHD in the clinic

Methods

Results

Conclusion