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

Abstract

Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.

Increasing Gene Editing Efficiency via CRISPR/Cas9- or Cas12a-Mediated Knock-In in Primary Human T Cells

“…Nucleotide polymorphisms in the target region can also affect editing [76,77]. One study performed on primary T cells reported that combined data on the epigenetic profiles of T cells and in silico gRNA prediction provided a much more accurate estimate of gRNA potency [78].…”

“…To increase the level of editing, several studies have proposed using two or three gRNAs per gene. The gain is not usually substantial compared to the most efficient gRNA alone [82][83][84], although it may be advantageous for epigenetically closed loci [78]. Furthermore, multiple DSBs are known to increase the likelihood of large chromosomal rearrangements, and thus this method of increasing the level of editing is not the most reliable [53,85,86].…”

Gene editing technology to improve antitumor T-cell functions in adoptive immunotherapy

“…Epigenetic architecture has been identified as one of those factors, as heterochromatin states impede Cas9 binding to the target region and inhibit DNA cleavage [ 69 , 70 ]. We have recently shown that the chromatin accessibility of T cells, as measured by ATAC sequencing, supports the selection of gRNAs that allow efficient gene editing in human T cells [ 71 ]. The combination of currently available prediction tools with epigenetic information has enabled precise selection of optimal gRNA targets.…”