Fig 1.
CRISPR/Cas9 RNP editing of human CD34+ hematopoietic stem and progenitor cells.
(A) Overview of sgRNA:Cas9 RNP nucleofection procedure. (B) & (C) Optimization of the Lonza nucleofection program selection for CD34+ HSPCs by introducing chemically modified mCherry RNA. Levels of mCherry expression were quantified 2-days post-nucleofection by flow cytometry (n = 1). (D) Comparing the optimized and original Lonza nucleofection program by targeting CD8 in CD34+ HSPCs from three unique donors with dual sgRNAs, 50 pmol each with Cas9 in a 2:1 ratio. Three days post-nucleofection gDNA was isolated and CRISPR activity quantified by Sanger sequencing and ICE analysis (n = 3, p-value = 0.0017, the range is included in the table). (E) Representative indel size distribution plots for the data presented in D. (F) Use of the optimized protocol to target CD8 in CD34+ HSPCs from three separate human donors (n = 8). Three days post-nucleofection gDNA was isolated and CRISPR activity quantified by Sanger sequencing and ICE analysis.
Fig 2.
Simultaneous CRISPR/Cas9 RNP editing of multiple genes in human CD34+ hematopoietic stem and progenitor cells and in vitro biological validation of editing.
(A) Optimization of input sgRNA amounts when nucleofecting dual sgRNAs for gene knockout. The listed pmole amounts were added for each sgRNA in combination with Cas9 in a 2:1 molar ratio. Three days post-nucleofection, gDNA was isolated and CRISPR activity quantified by Sanger sequencing and ICE analysis (n = 1). (B) Indel size distribution plots for the data presented in A illustrating the loss of the genomic fragment deletion with suboptimal editing efficiency. (C) Simultaneous triple gene KO by sgRNA:Cas9 RNP nucleofection. CD34+ cells were nucleofected with a pool of RNPs targeting the three YTHDF RNA binding proteins. Each pool contained 25 pmol each of 6 sgRNAs (2 sgRNAs per gene) with Cas9 in a 2:1 ratio. Three days post-nucleofection gDNA was isolated and CRISPR activity quantified by Sanger sequencing and ICE analysis. (n = 1) (D) Representative flow cytometry results of METTL3 KO by CRISPR/Cas9 RNPs recapitulating the in vitro erythroid differentiation phenotype previously reported by lentiviral shRNA knockdown of METTL3 [32]. Cells were assayed by FACS 7–9 days post-nucleofection. All viable cells from the nucleofection are shown. The control cells were KO for CD8 (n = 1). (E) TP53 KO by sgRNA:Cas9 RNP nucleofection. CD34+ cells from three independent donors were nucleofected with dual sgRNAs, 50 pmol each with Cas9 in a 2:1 ratio, targeting TP53. Three days post-nucleofection the cells were given 4 gray of ionizing radiation followed by isolation of protein extracts 3 hours later. TP53 protein levels were measured by Western blot with beta-actin as a loading control (n = 3). Relative levels of TP53 normalized to beta-Actin are plotted below.
Fig 3.
High efficiency in vivo gene KO following transplantation of CRISPR/Cas9 RNP edited human CD34+ cell into the MISTRG humanized mouse model.
(A) The optimized workflow for transplantation of CRISPR/Cas9 RNP edited CD34+ human cells into MISTRG mice. (B) Frequency of human CD45+ cells in the blood of MISTRG mice 8-weeks after transplantation of fetal CD34+ cells, treated either with mock nucleofection (n = 7) or METTL3 RNP (n = 8). (C) Frequency of hCD45+ cells in the blood of MISTRG mice after transplantation of mock nucleofected (n = 6) or CD33 RNP CD34+ cells (n = 9). (D) Lineage differentiation in the blood of the same mice. (E) Expression levels of cell surface CD33 by cells of each lineage in the blood. (F) Frequency of hCD45+ cells in the BM of MISTRG mice. (G) Lineage differentiation in the BM. (H) Expression levels of cell surface CD33 by cells of each lineage in the blood. (I) IHC identifying human CD3+ T cells, CD20+ B cells and CD163+ myeloid cells, and expression of CD33 in the spleen of MISTRG mice. (J) IHC identifying human CD163+ myeloid cells and expression of CD33 in the lung and liver.
Fig 4.
CD33 deficiency confers resistance to the CD33-targeting antibody-drug conjugate Mylotarg.
Frequency of monocytes (A, B), granulocytes (C) and other myeloid cells (D) in the blood (A) or BM (B-D) of mock nucleofected control and CD33 RNP mice, after treatment with Mylotarg. (E) Identification of CD163+ myeloid cells and expression of CD33 in the spleen of the same mice.