Fast and Efficient Gene Editing in Human Hematopoietic Cells

Although CRISPR/Cas9 is now accessible to a wide variety of cell-types and model systems, efficient editing of hematopoietic cells remains challenging. We have designed and optimized a protocol for rapid and efficient delivery of CRISPR/Cas9 to hematopoietic cell lines and primary cells. Combining electroporation's high transfection efficiency and the reduced cellular toxicity of Cas9 protein versus plasmid in suspension cells, we are able to produce highly efficient gene disruption and knock-in in a variety of human cell types, including acute myeloid leukemia (AML) cell lines, B-acute lymphoid leukemia (ALL) cell lines, primary T-lymphocytes and primary hematopoietic stem/progenitor cells (HSPCs). Our protocol involves rapid sgRNA template design and PCR amplification, followed by overnight in-vitro transcription, sgRNA purification and sgRNA-Cas9 ribonucleoprotein (RNP) formation. We began by testing the protocol on three AML cell lines, in which we observed up to 98% knock-out (KO) of the ubiquitous hematopoietic marker CD45 (%CD45^neg cells by flow cytometry: HL-60 - 98%, OCI-AML2 - 92%, Kasumi - 87%). Using multiple guides, we also induced KO of two B-cell markers (CD19 and CD22) in three B-cell cancer cell lines (BV173, Daudi and Nalm-6). In these three cell lines, up to 70% of cells displayed combined loss of both cell surface receptors, indicating disruption of all four alleles (%CD19^negCD22^neg cells by flow cytometry: BV-173 - 58%, Nalm-6 - 70%, Daudi - 18%).

We then optimized our editing strategy in human primary cells. We observed highly efficient CD45 loss (86±2%; n=3) in activated T-cells by flow cytometry and confirmed this KO frequency using high-throughput sequencing. We next measured CD45 gene disruption in CD34+ HSPC cells isolated from cord blood and found that our system had 75±10% editing efficiency (n=4). Importantly, a 48-hour period of cytokine stimulation with SCF/TPO/FLT3L prior to electroporation was required for efficient gene knockout (0hr: 8±4%, 24hr: 41±12%, 48hr: 73±16%; p^0vs24=0.0002, p^24vs48=0.003; n=8). Our protocol induced efficient gene disruption of several relevant targets in CD34+ cells including DNMT3A ex7 (69±4%; n=5), DNMT3A ex10 (86±14%; n=10) and NR3C1 (75±6%; n=5), and near complete loss of protein by western blot. To verify that the edited CD34+ HSPCs cells maintained engraftment and multilineage differentiation capacity, we transplanted Cas9 only (n=8) and Cas9/hCD45-sg1 RNP edited cells (n=8) into sub-lethally irradiated NOD scid gamma (NSG) mice. To avoid possible donor-dependent bias, each experimental pair (i.e. one Cas9 only replicate and one Cas9/hCD45-sg1 RNP treated replicate) was performed on cells derived from a single cord blood. Human cells successfully engrafted in the bone marrow of 16/16 recipients and spleens of 13/16 recipients. Importantly, we observed significant levels of engraftment by hCD45^neg cells in the bone marrow of 7/8 mice and in the spleen of 5/8 mice transplanted with Cas9/hCD45-sg1 RNP edited cells (Figure 1A; Figure 1B shows one representative pair). High-throughput sequencing confirmed that engrafted human cells in BM displayed hCD45indel frequencies consistent with the flow cytometry data.

Finally, we considered whether these editing strategies could be used to introduce specific point mutations into primary human HSPCs using Cas9-mediated homology directed repair (HDR). Single-stranded oligonucleotide HDR templates (ssODNs) with 90bp homology arms to the human CD45 locus were designed to introduce three basepair changes, two of which result in the generation of a BsiWI site near the CD45-sg1 cut site. High-throughput sequencing of treated human HSPC samples revealed efficient precise knock-in (22±4%; n=4) of the mutant allele.

In conclusion, we describe a fast and efficient protocol for both gene disruption and targeted gene editing of human hematopoietic cells, including HSPCs, using the CRISPR/Cas9 system. The ability to quickly and efficiently edit primary human HSPCs using HDR makes it possible to introduce or repair genetic variants identified in association with hematologic diseases such as leukemia or bone marrow failure. Moreover, the high efficiency of this system offers the possibility to perform large-scale combinatorial gene editing in HSPCs to model complex mutational landscapes.

Figure 1

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Figure 1

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