Journal: Nature medicine
Article Title: Highly efficient therapeutic gene editing of human hematopoietic stem cells
doi: 10.1038/s41591-019-0401-y
Figure Lengend Snippet: a , Eight modified synthetic (MS) sgRNAs targeting BCL11A enhancer DHS h+58 functional core marked with blue arrows. GATA and Half E-box motifs marked respectively with red or green. b , Editing efficiency of Cas9 coupled with various sgRNAs (each targeting BCL11A enhancer with exception of AAVS1 ) in CD34 + HSPCs measured by TIDE analysis. c , β-like globin expression by RT-qPCR analysis in erythroid cells in vitro differentiated from RNP edited CD34 + HSPCs. d , Correlation of BCL11A mRNA expression determined by RT-qPCR versus HbF by HPLC. Black dots represent samples edited with Cas9 coupled with different sgRNAs. The Pearson correlation coefficient ( r ) is shown. e, Editing efficiency as measured by TIDE analysis of Cas9:sgRNA RNP targeting AAVS1 or BCL11A DHS h+58 functional core (Enh) with MS-sgRNA-1617 in CD34 + HSPCs from β-thalassemia patients or healthy donors (β A β A ) of indicated β-globin genotypes. f-h , β-like globin expression by RT-qPCR normalized by α-globin ( P = 0.00017 for BCL11A enhancer as compared to AAVS1 edited for all comparisons as determined by unpaired two-tailed Student’s t tests), and HbF induction by HPLC analysis in erythroid cells in vitro differentiated. i , Enucleation of in vitro differentiated erythroid cells. j , Cell size measured by relative forward scatter intensity. k , Representative microscopy image showing rounder and more uniform appearance of enucleated erythroid cells following BCL11A enhancer editing. Blue arrow indicates poikilocytes. Bar = 15 μm. l , m , Imaging flow cytometry was used to establish a circularity index (l) and then quantify (m) circularity of enucleated erythroid cells. Bar = 5 μm. In all panels, data are plotted as mean ± SD and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.
Article Snippet: The modified synthetic sgRNA (2’-O-methyl 3’ phosphorothioate modifications in the first and last 3 nucleotides) was from Synthego. sgRNA concentration is calculated using the full-length product reporting method, which is 3-fold lower than the OD reporting method.
Techniques: Modification, Functional Assay, Expressing, Quantitative RT-PCR, In Vitro, Two Tailed Test, Microscopy, Imaging, Flow Cytometry
