CRISPR/Cas9 genome editing in human hematopoietic stem cells - PubMed (original) (raw)

CRISPR/Cas9 genome editing in human hematopoietic stem cells

Rasmus O Bak et al. Nat Protoc. 2018 Feb.

Abstract

Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper.

Figures

Figure 1

Figure 1

Schematic overviews of design strategies for different donor types. (a) A reporter gene expression cassette driving the expression of, for example, a fluorescent protein (FP) can be integrated site-specifically by homologous recombination. 400-bp homology arms (in gray) are split at the CRISPR/Cas9 cut site between nucleotides 17 and 18 of the sgRNA target site (target site depicted in white and PAM in red) and flank the transgene expression cassette (in blue). Upon HR, the cassette is integrated seamlessly into the cut site. (b) SNPs can be introduced (X → Y mutation depicted, blue and green, respectively) using a vector design with 1.2-kb homology arms that flank the region between the desired site of the SNP and the CRISPR/Cas9 cut site. In the donor, the region between the mutation and the cut site should be mutated (the example uses synonymous mutations denoted by asterisks; note that encoded amino acids are listed above nucleotides) to avoid early termination of the HR process due to full sequence homology. This also introduces necessary mutations to the PAM and sgRNA target site to prevent Cas9 recutting and INDEL formation after HR. (c) A cDNA sequence (in green, diverged using synonymous mutations, e.g., as depicted in b) can be introduced directly into the start codon (ATG, purple) of a gene to express a desired cDNA from the endogenously regulated expression elements. A separate expression cassette (blue) can be included after the cDNA, encoding, for example, a fluorescent protein (FP), which allows tracking and/or enrichment of targeted cells. The cDNA and reporter cassette are flanked by two 400-bp homology arms (gray) that flank the start codon and the CRISPR/Cas9 cut site. Seamless HR ensures that the cDNA is integrated in frame with the start codon. In an analogous manner, a 2A-cDNA cassette can be integrated immediately before the stop codon to link expression of a transgene to the expression of an endogenous gene. FP, fluorescent protein; LHA, left homology arm; pA, polyadenylation signal; RHA, right homology arm; SNP, single-nucleotide polymorphism.

Figure 2

Figure 2

Enrichment of gene-targeted CD34 + HSPCs using CRISPR/Cas9, AAV6, and FACS methodologies. (Left) Representative CD34 + HSPC FACS plots from day 4 post electroporation of Cas9 RNP and transduction of AAV6 (top) and transduction of AAV6 only (bottom) are shown, highlighting the generation of a reporterhigh (GFPhigh, shown in the red gate) population after the addition of Cas9 RNP (see also Supplementary Figure 1 for FACS plots that include staining for CD34 expression). At day 4 post electroporation, targeted HSPCs from GFPhigh (red), GFPlow (green), and GFPneg (blue) fractions were sorted and cultured for 15 d while monitoring GFP expression by flow cytometry every 3 d (right). Note that the reporterhigh population is > 96% reporter + after 15 d in culture, highly indicative that this population is enriched for stable integration of the reporter cassette. neg, negative; SSC, side scatter. Image adapted with permission from ref. , Springer Nature.

Figure 3

Figure 3

‘In–Out’ PCR strategy for genotyping on-target integration events in methylcellulose-derived colonies. (a) Schematic outlining the ‘In–Out’ PCR strategy for identifying on-target integration events in HSPCs. (Top) Primer design for the nontargeted allele using one primer that binds outside the left homology arm (LHA), and another primer that binds inside the right homology arm (RHA). Primers are depicted as red arrows. In the example presented, the PCR strategy will produce an 800-bp product (red) from a wild-type (WT)/INDEL allele. Note that the molecular weight of this band could be smaller or larger if an INDEL of substantial size is present. (Bottom) A targeted allele with a reporter cassette after CRISPR/Cas9 and AAV6-mediated homologous recombination. By using the same outside LHA (Out) primer as above, but with a reporter cassette–specific inside primer (In), this ‘In–Out’ PCR strategy will generate a 600-bp on-target integration-specific PCR product (purple). Primers are depicted as purple arrows. (b) A schematic representation of an agarose gel image showing the types of clonal integration events (when targeting an autosomal gene with one allele on each chromosome): WT or INDEL (800 bp, red), biallelic HR (600 bp, purple), and monoallelic HR (800 and 600 bp). Note that the presented PCR strategy is a three-primer PCR that analyzes all events in the same PCR. It is possible to separate the strategy into two PCR reactions. Furthermore, it is recommended to perform the same ‘In–Out’ strategy at the 3′ end of the integration and, importantly, to Sanger-sequence PCR bands to confirm seamless HR at both ends.

Figure 4

Figure 4

Gating strategy for engraftment analysis of NSG mice transplanted with targeted CD34 + HSPCs. Representative FACS plots showing gating scheme for identifying human cells (huCD45/HLA-ABC double positive) in the bone marrow of transplanted mice analyzed on a FACS Aria system with data analysis using the FlowJo software. (a) Representative FACS plot from the analysis of the bone marrow of a mouse not transplanted with human cells. The position of a human population is depicted in the red gate, which is transferred from b. The frequency of false-positive or contaminating human cells in this example is 0.001%. (b) Representative FACS plot from the analysis of bone marrow from a mouse transplanted with mock-electroporated cells. The left plot shows engraftment rates (2.2%) with human cells entailed in the red gate (positive for both huCD45 and HLA-ABC). The right plot shows GFP fluorescence of the human cells. A GFP + gate is shown in green. (c) Representative FACS plot from the analysis of bone marrow from a mouse transplanted with RNP + AAV-treated cells (bulk). Human engraftment (red gate) and the frequency of targeted cells (GFP +, green gate) in the total human population are gated as in a and b. Furthermore, the bilineage potential (lymphoid and myeloid) of the engrafted human cells is analyzed by the presence of B cells (CD19 +, lymphoid, blue gate) and myeloid cells (CD33 +, purple gate), within which the frequency of targeted cells (GFP + ) can also be quantified. (d) Representative FACS plot from the analysis of bone marrow from a mouse transplanted with RNP + AAV-treated cells sorted for GFPhigh expression immediately before transplantation. The gating scheme is identical to that described above for c. See Supplementary Figure 3 for the gating strategy upstream to the shown FACS plots. The experimental protocol was approved by Stanford University’s Administrative Panel on Laboratory Animal Care. Image adapted with permission from ref. , Springer Nature.

Figure 5

Figure 5

Flowchart for genome editing of CD34 + HSPCs and analysis of in vitro and in vivo HR frequencies. CD34 + cells are purified from fresh hematopoietic tissues or thawed from frozen stocks, and then stimulated with cytokines to preserve stemness, and promote cycling, and survival in vitro. Stimulated CD34 + HSPCs are then targeted using CRISPR/Cas9 and AAV6, HR efficiencies are evaluated in vitro, and CFU assays and NSG repopulation studies are initiated. Finally, in vivo HR frequencies in HSCs are evaluated by analysis of long-term engraftment of human cells in the bone marrow of NSG mice. Note that secondary transplants are recommended to confirm HR in true long-term HSCs.

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