Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

Abstract

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.

Figure 1. Targeting clinically relevant loci in human cells using CRISPR/Cas9

A) Schematic of gRNAs targeting B2M. B) Histogram of B2M surface expression in HEK293T cells. C) B2M deletion efficiency with various gRNAs in HEK293T cells; n=3 (mean±SEM). D) Schematic of gRNAs targeting CCR5. Orange and green arrows represent primer pairs used to amplify the region for analysis. E) Surveyor assay of each gRNA targeting CCR5 in K562 cells. % InDels is indicated under each guide. F) B2M deletion efficiency of selected gRNAs in primary CD4⁺ T cells in comparison to 293T cells; n=6 (mean±SEM). G) Surveyor assay of crCCR5_A and crCCR5_B targeting CCR5 in K562 cells and HSPCs. H) Clonal deletion efficiency of crCCR5_A and crCCR5_B targeting of CCR5 in HSPCs (n=2) as determined by Sanger sequencing. (Note: crB2M_14 is not depicted in panel A schematic, as it is located 20 Kb downstream of coding sequence.). See also Figure S1.

Figure 2. A dual gRNA approach for CRISPR/Cas9 genome editing in primary human hematopoietic stem and effector cells

A) Schematic of dual gRNA approach for targeting the B2M locus. gRNA pairs are in red. The offset in base pairs between Cas9 sites for each gRNA combination (right panel). B) B2M deletion efficiency in CD4⁺ T cells for 6 dual gRNA combinations (n=3; mean±SEM). C) FACS plots showing loss of B2M expression of either crB2M_13 or crB2M_8 alone or in combination in primary CD4⁺ T cells. D) Schematic of dual gRNA approach for targeting CCR5. gRNA pairs are shown in red. Orange and green arrowheads represent primer pairs used to amplify the region. The offset between the Cas9 sites of each gRNA pair (right panel). E) Gel electrophoresis image of CD34⁺ HSPCs derived clones targeted with crCCR5_D+Q analyzed by PCR. Note the deletion of the 205 bp region between the two gRNA cutting sites (top panel; WT: wild type; ΔCCR5: deleted; green * = WT; orange * = heterozygote; and red * = null clone). Clonal deletion efficiency for three dual gRNA combinations targeting CCR5 in CD34⁺ HSPCs (n=4; % mean±SEM; bottom panel). See also Figure S2.

Figure 3. CCR5-edited CD34⁺ HSPCs retain multi-lineage potential

A) Representative pictures of colonies formed in methylcellulose CFC assay (left panel) with quantified data on colony number and types are presented (right panel). Representative FACS plot showing human hematopoietic cell (hCD45⁺) engraftment and multi-lineage reconstitution at 12 weeks post-transplantation in the bone marrow (B) and spleen (C) of NSG recipient mice. D) PCR results confirmed predicted deletion of targeted region at CCR5 locus in human hematopoietic cells sorted from NSG mice transplanted with CRISPR/Cas9-treated HSPCs. PBMC (human peripheral blood mononuclear cells) from healthy donor taken as control. (WT: wild type, ΔCCR5: deleted)

Figure 4. Targeted capture and extremely deep sequencing of on-target and predicted off-target sites in CD34⁺ HSPCs

A) Schematic of targeted capture deep sequencing of on-target and predicted off-target sites (red bar); probe sets are indicated in blue. A 500 bp region flanking the site (in yellow) was included for detection of structural rearrangements (i.e. translocations). B) Plots showing sequencing depth coverage at both on-target (left panel) and off-target (right panel) sites, achieving a coverage exceeding 3,000x for all on-target sites. Decrease in sequencing depth at the on-target sites in dual-gRNA libraries is marked by arrow, supporting predicted deletions (bottom left; i=35 bp, ii=205 bp, iii=205 bp). C) Precise estimation of on-target mutation allele frequencies by capture sequencing. Notably, the rate of mutation exceeds previous estimates by PCR of predictable deletions, as smaller InDels and inversions also occur at appreciable frequencies. D) Estimation of mutation frequencies at predicted off-target sites (*One off-target site was statistically different from controls following correction for multiple comparisons; p ≤ 7.6×10⁻¹¹). N-fold enrichment is determined based on the ratio of non-reference reads in treated libraries compared to untreated library and represents the average of all off-target sites for a given experiment. Enrichment of 1 is equivalent to untreated control. **For reference to on-target enrichments, on-target combined represents the proportion of non-reference reads (including single and dual-gRNA treatments using a given gRNA) to total reads at on-target sites in treatment compared to control. See also Tables S1–S5.