CRISPR prime editing (PE) is an evolution of CRISPR-Cas9 gene editing, allowing precision editing of T cell DNA with high versatility and accuracy. In previous work, we reported efficient PE in primary human T cells (Petri, Zhang, Ma, Schmidts, et al., Nature Biotechnology 2021). However, knowledge regarding PE efficiency in T cell subsets remains limited. Here, we assessed PE in primary human CD4 and CD8 T cells.

We acquired synthetic prime editing guide RNAs (pegRNAs) with stability-increasing modifications and produced PE mRNA through in vitro transcription. We sorted primary human CD4 and CD8 T cells and activated them with CD3/CD28 magnetic beads. We nucleofected pegRNAs and PE mRNA and incubated cells for 72h. We extracted genomic DNA and amplified target loci. We then assessed editing efficiency using sequencing.

We measured PE activity in multiple donors at two target sites. PE enabled the modification of CD4 and CD8 T cells with up to 40 % efficiency, installing G > T and G > C substitution edits that are difficult to install with conventional CRISPR-Cas9 and CRISPR base editing.

PE of T cell subsets might allow the production of differentially edited CAR-T subset mixtures with potentially enhanced properties. Differentially-edited CAR-T subsets could be deployed at varying ratios depending on tumor type and employed CAR. Here, we show efficient PE in CD4 and CD8 T cell subsets, potentially enabling the generation of differentially prime-edited CAR-T subset mixes with enhanced properties.