Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells

Area of Science:

• Immunology
• Molecular Biology
• Gene Editing Technologies

Background:

• Adoptive immunotherapy utilizes genetically modified T cells to enhance anti-tumor functions.
• CRISPR/Cas9 technology allows gene knockout in T cells for therapeutic purposes.
• Efficient selection of guide RNAs (gRNAs) is crucial but challenging for gene ablation.

Purpose of the Study:

• To develop a strategy for selecting optimal gRNAs for CRISPR/Cas9 gene editing in human T cells.
• To evaluate the performance of existing in silico prediction tools in human T cells.
• To improve gene-editing efficiency in T cells for adoptive immunotherapy.

Main Methods:

• Collected pooled data on CRISPR/Cas9-mediated gene knockout in human T cells.
• Utilized transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) for epigenetic profiling.
• Integrated epigenetic data with sequence-based prediction tools to enhance gRNA selection.
• Investigated targeting epigenetically closed regions using dual gRNAs.
• Assessed the effect of IL-7 pretreatment on gene-editing efficiency in unstimulated T cells.

Main Results:

• Existing in silico tools alone were insufficient for accurate prediction of indel percentages in T cells.
• Combining epigenetic profiles (ATAC-seq) with sequence-based predictions significantly improved gene-editing efficiency.
• Epigenetically closed regions could be targeted effectively by designing adjacent gRNAs.
• Pretreatment with IL-7 enhanced the gene-editing efficiency of unstimulated T cells.

Conclusions:

• A novel strategy combining epigenetic information and sequence-based tools enhances gRNA selection for CRISPR/Cas9 in human T cells.
• This approach overcomes limitations of current prediction tools and enables efficient gene editing.
• The findings facilitate improved T-cell engineering for adoptive immunotherapy and other therapeutic applications.