Person:

Ferreira, Leonardo Manuel Ramos

Human leukocyte antigens (HLA) are important determinants of self-nonself immune recognition. HLA-G, uniquely expressed in the placenta, is believed to be key to fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Using a Massively Parallel Reporter Assay (MPRA), we discovered a 121 bp sequence 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, deletion of Enhancer L using a CRISPR/Cas9 dual guide approach resulted in complete ablation of HLA-G expression in a trophoblast cell line. This finding was confirmed in primary extravillous trophoblasts isolated from human placenta. RNA-seq analysis demonstrated that Enhancer L regulates HLA-G expression specifically. Moreover, DNase-seq and Chromatin Conformation Capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. GATA2, GATA3, and CEBPB, factors essential for placentation, associate with Enhancer L and regulate HLA-G expression levels. These results establish long-range chromatin looping as a novel mechanism controlling trophoblast-specific HLA-G expression at the maternal-fetal interface.

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.