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.
Genome engineering is not only a powerful research tool, it is also being developed to cure human diseases, including those of the blood and immune system, most of which can be categorized as still having a great unmet medical need1,2,3,4. Ex vivo–engineered nuclease-mediated gene editing by HR in hematopoietic stem and progenitor cells (HSPCs) can shed light on stem cell gene function through precise genetic manipulations, and can potentially define a curative strategy for currently incurable hematological diseases. The RNA-guided Type II CRISPR/Cas9 genome-editing system uses a single protein, Cas9, that is guided by a chimeric single-guide RNA (sgRNA) to target DNA through Watson–Crick base-pairing. Because of its simplicity and robustness, it is becoming the most widely used engineered nuclease for editing of mammalian genomes5,6. We have previously shown that the high editing performance of a plasmid-based CRISPR/Cas9 system in cell lines did not translate into high editing activity in primary cell types such as human primary T cells and HSPCs7. Protection of both sgRNA termini with chemically modified nucleotides increases sgRNA stability and renders the 'all RNA'-based CRISPR/Cas9 system highly effective in primary HSPCs and T cells7. In addition, ribonucleoprotein (RNP) delivery of Cas9 precomplexed with chemically modified sgRNAs consistently increased activity in T cells7,8 and CD34+ HSPCs (R.O.B., D.P.D., and M.H.P., data not shown). Multiple publications have also shown efficient genome editing in T cells9 and HSPCs10,11 without modified sgRNAs in the context of Cas9 RNP delivery. In these studies, however, a direct comparison with synthetic sgRNAs with modifications was not performed, and it remains possible that the generally most active form of an sgRNA, even in the context of RNP delivery, is one that is synthetically manufactured with end modifications protecting against endogenous exonuclease degradation and innate immune stimulation.
Creating a locus-specific double-strand break (DSB) with engineered nucleases forms the foundation of genome editing12,13. DSBs can be resolved by one of the two highly conserved competing repair mechanisms, nonhomologous end-joining (NHEJ) and HR14. NHEJ repair is the default pathway that functions throughout the cell cycle to repair breaks by ligation of DNA ends without end processing, sometimes resulting in small insertions or deletions (INDELs) at the site of the break. By contrast, HR is normally most active during the S or G2 phase of the cell cycle, when an undamaged sister chromatid is available to serve as an HR repair template. HR can be harnessed for creating precise DNA changes by supplying an exogenous DNA donor template, as long as the donor has homology arms that are identical to the region surrounding the DSB. This way, disease-causing single-nucleotide polymorphisms (SNPs) can be reverted or entire open reading frames can be inserted at specific genomic sites15 (Fig. 1). Although no studies using CRISPR and rAAV6 donors in CD34+ HSPCs have comprehensively investigated the impact of homology arm size and position relative to the nuclease cut site, studies using transcription activator–like effector nucleases (TALENs) and plasmid donors have found that maximal HR-mediated editing occurs when both the homology arms are at least 400 bp16.
Recombinant adeno-associated viruses (rAAVs) have been shown to naturally mediate high frequencies of HR in mammalian cells without stimulation of DSBs17,18,19. Wild-type AAVs are nonenveloped single-stranded DNA viruses that consist of an ∼4.7-kb genome encoding replication (rep) and capsid (cap) genes between 145-bp inverted terminal repeats (ITRs)19. Naturally occurring and engineered serotypes exist that have differential tropism, and serotype 6 has been shown to be the most efficient serotype for transduction of HSPCs and primary T cells20,21,22. AAVs are dependent upon adenoviruses (helper viruses) to replicate in cis; however, packaging of rAAV vectors with user-defined DNA cargo is possible by cotransfection of the following three types of plasmids into a host cell line such as HEK293T: (i) transfer plasmids with the homology arms and transgene of interest between the two ITRs, (ii) rep/cap-encoding plasmids (in this protocol rep2/cap6), and (iii) helper plasmids encoding the adenoviral helper proteins (in this protocol Ad5). As rAAVs can generate a high vector copy number in the nucleus and avoid innate immunity, they are ideal templates for HR following use of engineered nuclease-mediated DSBs in primary human cells. Accordingly, several recent reports have efficiently used rAAV6 as donor template for HR in primary human HSPCs21,22,23,24.
Human hematopoiesis is the process that generates all blood and immune cells from HSCs with self-renewing capacity25,26. Although progress has been made in identifying bona fide repopulating and self-renewing HSCs by immunophenotyping27,28, the CD34+ cell surface marker has been readily used to identify a heterogeneous population of HSPCs. This population generally contains ∼0.1–1% HSCs with long-term repopulation capacity (LT-HSCs). This capacity is normally experimentally tested in immunodeficient mice, e.g., in nonobese diabetic (NOD)-severe combined immunodeficiency (SCID)-gamma (NSG) mice, which lack innate and adaptive immunity to allow human cell engraftment in the mouse bone marrow29. Several reports have recently shown that HR is more efficient in progenitor cells, as compared with LT-HSCs10,15,30,31. Although current investigations attempt to augment HR rates in LT-HSCs, this is the biggest hurdle in accelerating clinical strategies for HR-based genome editing for blood and immune system disorders. We therefore devised a reporter-based enrichment paradigm that takes advantage of a log-fold-higher transgene expression after successful HR into the desired locus (compared with low AAV6 episomal expression), and this methodology could yield strikingly higher frequencies of modified cells in the transplanted mice (up to 97%)15. Thus, the enrichment methodology can solve the potential problem of inefficient HSC targeting.
In this protocol, we provide a reproducible methodology for achieving HR in HSPCs using CRISPR/Cas9 and rAAV6 homologous donor delivery. We describe in detail (i) sgRNA selection and AAV6 homologous donor design and construction, (ii) electroporation and transduction protocols, (iii) a flow cytometry–based strategy to enrich for a population of HPSCs with >90% targeted integration, and (iv) in vitro and in vivo assays for determining HR frequencies in HSPCs. We also describe the use of the protocol in primary human T cells (Box 1). This is a comprehensive protocol for targeting human HSPCs for HR to investigate hematopoietic gene function and disease modeling, as well as preclinical development of HSC-based cell and gene therapies.
Comparison with other technologies
Just like Cas9, other engineered nucleases, such as zinc-finger nucleases (ZFNs), TALENs, and hybrid meganuclease-TALENs (megaTALs), can stimulate DSBs in mammalian genomes. However, Cas9 has two distinct advantages as compared with these other designer nucleases in stimulating DSBs in HSPCs. First, ZFNs, TALENs, and megaTALs are more cumbersome to construct and require an extensive molecular biology skill set. By contrast, the CRISPR/Cas9 system from Streptococcus pyogenes uses a simple 20-nt guide sequence to facilitate a locus-specific DSB. Furthermore, the chimeric sgRNA can be chemically synthesized with modifications at the ends that make it highly efficient in primary human HSPCs. Second, recombinant Cas9 protein can be easily produced and precomplexed with sgRNAs on the bench and delivered by electroporation as RNP complexes, which shortens nuclease exposure and provides a hit-and-run mechanism that decreases potential unwanted off-target DSBs.
Although rAAV6 can serve as a homologous donor template, there are other donor template platforms that have been shown to mediate efficient HR in HSPCs; these include single-stranded oligonucleotides (ssODNs)10,31 and integration-defective lentiviral (IDLVs) vectors30. Although current literature may suggest that rAAV6 is a more efficient donor than IDLV21, a thorough comparison has yet to be performed. In comparison with ssODNs, which are usually ∼50–200 bp in total size and can therefore mediate only small genomic changes, rAAV6 donor templates can mediate precise SNPs, as well as insert transgene cassettes up to ∼4 kb in size if a single donor vector is used, or can insert even larger transgene cassettes if a sequential HR strategy with two donor vectors is used32. If single-point mutations are the desired genomic change, ssODNs may be a useful donor template for use in HSPCs, as they are easily produced and have been shown to work well in vitro10. However, ssODNs are too small to encompass an expression cassette for a reporter gene, and are therefore not compatible with the enrichment protocol for HSCs with precise targeted integration by flow cytometry that we outline in this protocol, which we believe is the key step for eliminating the greater number of nontargeted HSCs that outcompete targeted HSCs for engraftment in the host bone marrow.
Precise genome editing in HSCs has several advantages compared with conventional lentiviral vector (LV)-based gene transfer methods. First, genome editing maintains endogenous regulatory elements, preserving physiologic spatiotemporal regulation of gene expression1,3. Second, as LVs integrate semirandomly within the genome, the possibility always remains of insertional mutagenesis near or in oncogenes and/or tumor suppressor genes, confounding experimental results and possibly, although fortunately not yet described, leading to leukemogenesis in therapeutic settings. Third, semirandom lentiviral integration leads to expression heterogeneity among cells, which can be a major confounder in understanding gene-cell function in a heterogeneous population such as HSPCs. For gene therapy, this creates a population of cells with differential potency, potentially requiring higher cell doses or higher vector copy numbers. Fourth, LV-based gene addition does not allow targeted gene knockout by integration of a reporter expression cassette into the gene of interest, which enables tracking and enrichment of knockout cells. These reasons make genome editing the preferred genetic manipulation strategy to elucidate HSC gene function, as well as to correct disease-causing genetic mutations for HSC-based therapies.
Limitations of the protocol
One of the main limitations of the CRISPR/Cas9 system is the need for a protospacer adjacent motif (PAM) sequence within the gene of interest; however, the 5′-NGG-′3 for SpCas9 can on average be found every 8–12 bp in the human genome and thus does not usually hinder application33. Cas9 variants have been engineered with other PAM specificities that might circumvent this problem, although these engineered variants have not as yet been shown to mediate high levels of editing in primary human cells34,35. In addition, appropriate donor design (Supplementary Methods), as described in this protocol, can also usually solve the problem when the CRISPR/Cas9 nuclease site is at a distance from the desired change. A limitation of our enrichment methodology is the need for an exogenous promoter driving the reporter transgene for enrichment (such as GFP or truncated nerve growth factor receptor (tNGFR)) for purifying targeted HSPCs early in the manufacturing process via flow sorting. We do note that the enhanced transgene expression after HR seems to be independent of the chosen promoter, which would allow the use of promoters with varying strengths in HSPCs or inducible promoters. Furthermore, it is possible that active endogenous promoters can be used to drive expression from enrichment cassettes for purifying targeted HSPCs. Nevertheless, the enrichment scheme is fundamental in removing nontargeted HSCs that can outcompete targeted HSCs for repopulation in the host bone marrow.
sgRNA design. Excellent protocols for designing and testing sgRNAs have been described previously33. Here, we outline the key steps for identifying and characterizing a highly active locus-specific sgRNA, which is key to achieving optimal HR frequencies in HSPCs (Supplementary Methods). In brief, we routinely screen four to eight sgRNAs (depending on the available PAM sites in the targeted region) in the immortalized K562 cell line. Once a potent candidate sgRNA has been identified, a chemically modified synthetic sgRNA is ordered (Reagents section) from TriLink Biotechnologies (1-μmole minimal synthesis) or Synthego (3-nmole minimal synthesis; as new commercial sources for full-length synthetic modified sgRNAs become available, these might be used as well) and is functionally validated in human CD34+ HSPCs. Alternatively, we routinely bypass the functional screen in K562 cells and directly screen a panel of chemically modified synthetic sgRNAs in CD34+ HSPCs. Once high activity (as measured by the frequency of insertions and deletions using tracking of INDELs by decomposition (TIDE) analysis) of an sgRNA in HSPCs has been confirmed, a homologous donor template to introduce a desired genomic change is designed and cloned into an AAV vector plasmid. In general, the CRISPR cut site (between base pairs 17 and 18 of the 20-nt complementary sequence of the sgRNA) should ideally be located as close to the intended genomic change as possible36, although with proper design of the donor vector, we have observed substantial activity at a distance of 40 bp from the cut site15. In our experience, the farther the homology arms from the break site, the lower the HR efficiencies. When adding transgene expression cassettes into a safe harbor locus, the location of the sgRNA is less restricted, but if adding full cDNA cassettes to be driven by the correct endogenous regulatory context, e.g., directly into the start codon of a gene, the sgRNA target site should be located as close to the intended integration site as possible.
Donor design. The standard HR donor design is two homology arms (symmetric in length) that flank the transgene expression cassette or the mutations that will be introduced (Fig. 1a; Supplementary Methods). Homologous DNA donor design has been well described in excellent protocols referenced here37,38. Here, we will describe, in brief, the key concepts to consider when designing an AAV6 homologous DNA donor for CRISPR/Cas9-stimulated HR in HSPCs. Furthermore, details outlining the cloning of HR donors into an AAV plasmid backbone, and the production and purification of AAV vector particles are presented in the Supplementary Methods. The AAV packaging capacity of 4.7 kb limits the size of the donor. The ITRs and homology arms are indispensable elements of the vector, and the ITRs are combined at ∼270 bp. We recommend homology arms no <400 bp each, and we have not observed any substantial advantage of extending the length16, nor any advantage to using self-complementary AAV6 (double-stranded) over single-stranded AAV6 templates (R.O.B., D.P.D., and M.H.P., data not shown). This leaves ∼3.6 kb for the insert, so for HR of large transgenes or multicistronic cassettes, careful consideration must be made when designing this. For small genome modifications, such as introduction of SNPs, we recommend 1,200-bp homology arms to keep the total vector size >2.4 kb, which is greater than half of the AAV packaging capacity and prevents concatemer packaging39 (Fig. 1b). If the HR process is to integrate DNA exactly at the double-strand break, the arms should be split at the CRISPR cut site (Fig. 1a). If the desired modification is separate from the CRISPR cut site, the homology arms should flank the region between the cut site and the site of the modification (Fig. 1b,c). Two important considerations must be made in this situation: (i) If the target site is not disrupted by the introduced modification, Cas9 might introduce an INDEL after HR has taken place. To avoid this, mutations must be introduced (synonymous, if necessary) in the PAM or the sgRNA target site of the donors40 (Fig. 1b). (ii) If there is sequence homology between the donor and the target DNA in the region between the cut site and the introduced changes, termination of the HR process may occur due to the nascent DNA strand leaving the donor and annealing back to the chromosomal DNA in this region. To avoid this, mutations (synonymous, if necessary) should be introduced to minimize homology in the region between the cut site and the site of the desired change (Fig. 1b). Several studies have shown that mutations at codon wobble positions suffice to prevent premature HR termination15,41. If a cDNA cassette is to be integrated at the exact position after the start codon, the sgRNA target site should be located as close to the start codon as possible to minimize the gap between the start codon and the cut site, which, if too large, can negatively influence HR rates (Fig. 1c).
rAAV vector production. Excellent and comprehensive protocols have already described the production, purification, and titering of recombinant AAVs (rAAVs)38,42, and these provide sufficient resources to make rAAV6 (Supplementary Methods). We have detailed the critical steps for rAAV6 vector production and purification in the Supplementary Methods. In brief, rAAV vectors are commonly produced in HEK293T cells by the triple-transfection method, which involves transfection of (i) an ITR-containing transfer plasmid, (ii) a plasmid expressing the adenoviral proteins and RNAs required for helper functions, and (iii) a plasmid expressing the AAV rep and cap proteins that define the serotype. We use a transfer plasmid carrying ITRs from AAV2 (Reagents) and assemble the donor plasmid by standard Gibson assembly of the linear transfer plasmid backbone with terminal ITRs and PCR fragments containing the homology arms amplified from genomic DNA and the insert amplified from a desired expression plasmid. SNP donors can be made in a similar manner using standard site-directed mutagenesis techniques to introduce the SNPs. For AAV production, we use the dual-plasmid transfection system previously described38, in which the required adenoviral and AAV genes are combined on a single helper plasmid, pDGM6, which was described previously43 and can be obtained from the Russell lab at the University of Washington (Reagents). We routinely use rAAV vectors purified from iodixanol gradients for HR experiments in HSPCs, but note that other purification methods work as long as they produce pure rAAV preparations with a low proportion of empty capsids. A high-titer preparation is essential to achieving high transduction rates and avoiding toxicity induced by the dilution of the stem cell culture media when using high volumes of AAV vector. We recommend that the total volume of AAV not exceed 20% of the total culture volume (ideally, <10%). For titering the AAV preparations, we suggest using quantitative PCR on the ITRs to quantify the number of vector genomes as described previously44. It is important to note that the titer obtained is not a functional titer. A wide range of different multiplicities of infection (MOIs, i.e., vector genomes (vg) per cell) have been used in studies performing HR in HSPCs using AAV6. For example, Wang et al. used MOIs of 1–10 × 103 vg/cell21, whereas De Ravin et al. used MOIs of 1–3 × 106 vg/cell23. We believe that this large difference is due to differences in titering methodology and/or differences in AAV purity. We suggest that AAV titers be normalized to the AAV2 Reference Standard Material obtainable from the American-Type Culture Collection (ATCC, cat. no. VR-1616)45. This will allow more-streamlined titers, but will not address differences in AAV purity. We recommend that functional AAV titration be performed in HR experiments in HSPCs to identify the lowest MOI that yields maximum HR frequencies with minimal cellular toxicity.
Enriching cells that have undergone homologous recombination (Steps 47–52). An intrinsic feature of single-stranded AAV is that expression relies on second-strand synthesis, which subsequently produces dsDNA that is transcriptionally competent. We have recently reported on the observation that episomal AAV6 expression leads to low levels of reporter gene expression, even when driven from constitutive strong viral promoters in primary cells15. By contrast, we found that the reporter expression was increased more than a log-fold after HR-mediated chromosomal integration of the same expression cassette (Fig. 2; Supplementary Fig. 1). We have found that this phenomenon does not depend on cell type, target locus, exogenous promoter, or designer nuclease system used. The reporter shift is apparent as early as 24 h after electroporation and transduction (peaks at 3–4 d), and depends on transgene expression kinetics and proliferation status of the cells. We have used this shift in transgene expression to sort cells with reporterhigh expression and have obtained a purified targeted population with up to 99% purity. Importantly, we have shown in serial transplants that this enriched targeted population contains LT-HSCs.
Colony-forming unit assay and clonal genotyping (Steps 22–46). The colony-forming unit (CFU) assay is a progenitor assay that assesses the potential of progenitor cells to form colonies in semisolid methylcellulose media. This assay monitors the number of progenitor cells in the targeted population, as well as the proportion of lineage-committed progenitors (myeloid: CFU-GM; erythroid: BFU-E and CFU-E) and multilineage progenitors (mixed myeloid and erythroid: CFU-GEMM). It also enables the analyses of clonal genotypes by PCR screening of colonies for targeted integration of a transgene expression cassette. For this, an 'In–Out' PCR approach is used, in which one primer is located in the targeted genomic locus outside the region of the homology arm and the other primer is located inside the transgene cassette (Fig. 3a). Preferably, two PCRs are performed, identifying both the 5′ and the 3′ junction of the integration. If desired, a third primer can be included for identifying alleles without integration. This primer should be located in the genomic region on the opposite side of the sgRNA target site from the primer outside the homology arm (Fig. 3). It is critical that this primer be located at a distance of at least 50 bp away from the sgRNA cut site, as INDELs may otherwise disrupt the primer-binding site. HR frequencies for integration of SNPs can be assessed by droplet digital PCR or sequencing approaches (such as next-generation sequencing or TOPO cloning), or, if the genomic modifications generate a novel restriction site, restriction fragment length polymorphism (RFLP) analysis can quantify HR rates as described thoroughly before33. The same primer design as outlined above should be used with one primer located outside the region of the homology arms.
Repopulation assay in transplanted immunocompromised mice (Steps 53–65). Importantly, the CFU assay does not analyze the presence of HSCs with self-renewal and multilineage capacity, and for such verification, transplantation into immunocompromised mice is necessary. Although several mouse strains have been used for human hematopoietic repopulation, we recommend using the NSG strain, which is highly supportive of human engraftment and hematopoietic repopulation. If using other strains, more cells may need to be transplanted for robust engraftment. If transplanting bulk, nonenriched cells (RNP electroporation + AAV6 transduction) or RNP-electroporated CD34+ HSPCs without AAV6 donor transduction, transplantation can be performed as early as 2 h to 2 d after electroporation. HSPCs enriched for targeting via a reporter gene are transplanted directly after enrichment, when the frequency of reporter-positive cells peaks (Steps 17–21). Different standards for confirming long-term repopulating capacity of stem cells in NSG mice have been reported from 12–20 weeks in both primary and secondary recipients, respectively. Our recommendation is to assess engraftment 16 weeks after primary transplantation, and optionally perform secondary transplants and analyze these after 12 weeks to confirm true repopulating capacity.
Controls. As a positive control for Cas9/sgRNA activity in CD34+ HSPCs, we refer to the published sgRNA targeting the HBB gene and the matched HBB donor vector encoding GFP15. For all targeting experiments, a mock-electroporation control (no Cas9 RNP) is essential. This should be split into two wells: one that receives the AAV donor and one that does not. The latter is used for flow cytometric gating of reporter+ cells, and the former is used to set the reporterhigh gate (Fig. 2). Similarly, for the colony-forming unit assay and engraftment studies in NSG mice, a mock control will serve as a positive control and a potency reference for colony formation/distribution and engraftment.
HSPC cell culture
SFEM II (StemCell Technologies, cat. no. 9655)
IL6 (PeproTech, cat. no. 200-06)
SR1 (CellagenTech, cat. no. C7710)
UM171 (StemCell Technologies, cat. no. 72914)
Flt3L (PeproTech, cat. no. 300-19)
TPO (PeproTech, cat. no. 300-18)
SCF (PeproTech, cat. no. 300-07)
FBS (Fisher Scientific, cat. no. 10438026)
Penicillin–streptomycin (Fisher Scientific, cat. no. 15070063)
Fresh or frozen CD34+ human HSPCs (either acquired per institutional protocol or purchased from various commercial sources, including AllCells (CB-derived, cat. no. CB008F; mPB-derived, cat. no. mPB015F)). If available, investigators can purify CD34+ HSPCs, for example, from umbilical cord blood, bone marrow aspirates, or mobilized peripheral blood per institutionally approved protocols and following the FACS-based or immunomagnetic microbead technology protocols presented here46
Cas9 recombinant protein with nuclear localization signal (NLS; Life Technologies, cat. no. B25641 or IDT, cat. no. 1074182)
Synthetic sgRNA, identified and designed as described in the Supplementary Methods, with three terminal nucleotides modified with 2′-O-methyl phosphorothioate (bold in the sequence below). The 20-nt targeting region is indicated by Ns (TriLink, custom RNA synthesis, quote by request, or Synthego, synthetic sgRNA; example below).
K562 cell line (ATCC, cat. no. CCL-243)
ATP·disodium salt (Sigma-Aldrich, cat. no. A3377)
MgCl2·6H2O (Sigma-Aldrich, cat. no. M2393)
Nuclease-free ultrapure water
KH2PO4 (Sigma-Aldrich, cat. no. P5655)
NaHCO3 (Sigma-Aldrich, cat. no. S5761)
Glucose (Sigma-Aldrich, cat. no. G8270)
RPMI 1640 (GE, cat. no. SH30027.01)
Human T Cell Nucleofection Kit (Lonza, cat. no. VPA-1002)
P3 Primary Nucleofection Kit (Lonza, cat. no. PBP3-00675)
KCl (Moltox, cat. no. 26-645)
MgCl2 (Sigma-Aldrich, cat. no. M2393)
Na2HPO4/NaH2PO4, pH 7.2 (Boston BioProducts, cat. no. BB-180)
Mannitol (Sigma-Aldrich, cat. no. M4125)
Analysis of HR efficiencies
MethoCult Enriched (StemCell Technologies, cat. no. 04435)
Zero-Blunt TOPO Cloning Kit (Fisher Scientific, cat. no. K280020)
Propidium iodide at 1 mg/ml (Sigma-Aldrich, cat. no. P4864)
Immunodeficient NOD-SCID Il2rg/ (NSG) mice, preferably female to support the highest engraftment47, 6–8-weeks old (The Jackson Laboratory)
PBS, 1×, without calcium and magnesium (VWR, cat. no. 45000-448)
QuickExtract DNA Extraction Solution (Epicentre, cat. no. QE09050)
GeneJET Gel Extraction Kit (Fisher Scientific, cat. no. K0691)
Agarose (Fisher Scientific, cat. no. 17852)
50× TAE buffer (Bio Rad, cat. no. 1610773)
1 kb Plus DNA ladder (Fisher Scientific, cat. no. 10787018)
100-bp DNA ladder (Fisher Scientific, cat. no. SM0323)
Phusion Green Hot Start II High-Fidelity PCR Master Mix (Fisher Scientific, cat. no. F566S)
Midori Green Nucleic Acid Staining Solution (Bulldog Bio, cat. no. MG04)
Red blood cell lysis buffer, 1× (eBioscience, cat. no. 00-4333-57)
Dextran (Sigma-Aldrich, cat. no. 31392)
DNase (deoxyribonuclease I; Worthington Biochemical, cat. no. LS002007)
Heparin sodium (Fisher Scientific, cat. no. BP2425)
Ficoll-Paque Plus (GE, cat. no. 17-1440-03)
Trypan Blue (Corning, cat. no. 25-900-CL)
Antibodies (for immunophenotyping HSPCs or HSCs in vitro; GFP compatible)
Anti-human CD34-APC or CD34-APC-Cy7 (if using tNGFR as reporter), Clone 561 (BioLegend, cat. no. 343608 or 343614)
Antibodies (optional for staining for HSC phenotypic markers)
Anti-human CD45RA-BV605 Clone HI100 (BioLegend, cat. no. 304134)
Anti-human CD90-BV421 Clone 5E10 (BD, cat. no. 562556)
Anti-human CD38-PE-Cy7 Clone HIT2 (BD, cat. no. 560677)
Anti-human CD-123-PE (BD, cat. no. 554529)
Lineage antibodies (optional; for staining for HSC phenotypic markers):
Anti-human CD2-PE-Cy5 (BD, cat. no. 555328)
Anti-human CD3-PE-Cy5 (BD, cat. no. 555341)
Anti-human CD4-PE-Cy5 (BD, cat. no. 555348)
Anti-human CD8-PE-Cy5 (BD, cat. no. 555368)
Anti-human CD16-PE-Cy5 (BD, cat. no. 555408)
Anti-human CD19-PE-Cy5 (BD, cat. no. 555414)
Anti-human CD20-PE-Cy5 (BD, cat. no. 555624)
Anti-human CD56-PE-Cy5 (BD, cat. no. 555517)
Anti-human CD235a-PE-Cy5 (BD, cat. no. 559944)
Anti-human CD14-PerCP (BD, cat. no. 340585)
Antibodies (for human engraftment analysis in transplanted mice; GFP compatible)
Human TruStain FcX (BioLegend, cat. no. 422301)
Anti-human HLA-A/B/C-APC-Cy7 Clone W6/32 (BioLegend, cat. no. 311426)
Anti-human CD45-V450 Clone HI30 (BD, cat. no. 560368)
Anti-human CD33-PE Clone WM53 (BD, cat. no. 555450)
Anti-human CD19-APC Clone HIB19 (BD, cat. no. 555415)
Anti-mouse Ter119 PE-Cy5 Clone TER-119 (eBioscience, cat. no. 15-5921-82)
Anti-mouse CD45.1-PE-Cy7 Clone A20 (eBioScience, cat. no. 25-0453-82)
Antibodies (for immunophenotyping HSCs in vivo; GFP compatible)
Anti-human CD45-V450 Clone HI30 (BD, cat. no. 560368)
Anti-human CD10-APC-Cy7 Clone HI10a (BioLegend, cat. no. 312212)
Anti-human CD38-PE-Cy7 Clone HIT2 (BD, cat. no. 560677)
Anti-human CD34-APC Clone 8G12 (BD, cat. no. 340441)
Anti-mouse CD45.1-PE-Cy5 Clone A20 (eBioScience, cat. no. 15-0453-82)
Anti-human CD271 (tNGFR)-APC Clone ME20.4 (BioLegend, cat. no. 345108)
Anti-human CD34-FITC Clone 8G12 (BD, cat. no. 348053)
Steriflip 0.45-μM filter (Millipore, cat. no. SE1M003M00)
Electroporation cuvettes (if using Amaxa Nucleofector IIb; VWR, cat. no. 89047-208 or Fisher Scientific, cat. no. FB102)
Falcon FACS tubes with cell strainer (Corning, cat. no. 352235)
Cell strainers, 100 μm (Fisher Scientific, cat. no. 22-363-549)
Long transfer pipettes (Fisher Scientific, cat. no. 13-711-39)
BD FACS Sort II Aria (BD, cat. no. special order)
FlowJo software (FlowJo, https://www.flowjo.com/solutions/flowjo/downloads)
50-ml Falcon conical tubes (Fisher Scientific, cat. no. 352070)
15-ml Falcon conical tubes (Fisher Scientific, cat. no. 352096)
4D Nucleofector Core Unit and X Unit (Lonza, cat. nos. AAF-1002B and AAF-1002X)
Amaxa Nucleofector IIb (Lonza, cat. no. AAB-1001)
Sorvall Legend XTR Centrifuge (Fisher Scientific, cat. no. 75004521)
Faxitron Cabinet X-ray System (Faxitron, model no. 43855F)
Extra Fine Bonn Scissors (FST, cat. no. 14084-08)
Disposable scalpel blades, sterile, blade no. 22 (VWR, cat. no. 21909-626)
High-precision no. 4 style scalpel handle (Fisher Scientific, cat. no. 12-000-164)
Straight medium-point forceps (Fisher Scientific, cat. no. 16-100-106)
MACS CD34 MicroBead Kit, human (Milteneyi, cat. no. 130-046-702)
Heracell CO2 incubator capable of controlling oxygen concentration (Fisher Scientific, cat. no. 51026282)
C1000 Touch Thermal Cycler with Dual 48/48 Fast Reaction Module (Bio-Rad, cat. no. 1851148)
Thermo Scientific MaxQ 5000 shaking incubator (Fisher Scientific, cat. no. SHKE5000)
Precision general-purpose water bath (Fisher Scientific, cat. no. 2853)
Heating block (Fisher Scientific, cat. no. 88870002)
NanoDrop One (Fisher Scientific, cat. no. ND-ONE-W)
LightCycler 480 Instrument II (Roche, cat. no. 05015243001)
0.22-μm Vacuum filter (EMD Millipore, cat. no. SCGP00525)
K562 electroporation solutions
Solution I: Dissolve 2 g of ATP·disodium salt and 1.2 g of MgCl2·6H2O in 10 ml of nuclease-free ultrapure water, filter-sterilize, make 20-μl aliquots, and store them at −20 °C for up to 2 months. Solution II: Dissolve 6 g of KH2PO4, 0.6 g of NaHCO3, and 0.2 g of glucose in 500 ml of nuclease-free ultrapure water, adjust the pH to 7.4, filter-sterilize, make 1-ml aliquots, and store them at 4 °C for up to 3 months. Mix 20 μl of solution I with 1 ml of solution II. This makes enough K562 electroporation solution for ten electroporations (100 μl each).
1 M electroporation solution
The 1 M electroporation solution is 5 mM KCl, 15 mM MgCl2, 120 mM Na2HPO4/NaH2PO4, pH 7.2, and 50 mM mannitol. Store aliquots at −20 °C for up to 1 year48.
Human engraftment antibody cocktail
Prepare a master mix of the following antibody volumes for 1× samples: huCD45 (2 μl), CD33 (2 μl), mTer119 (0.25 μl), mCD45.1 (0.5 μl), CD19 (10 μl), HLA-A/B/C (1 μl), and if using tNGFR as reporter gene, include CD271 (5 μl). Store at 4 °C in the dark for up to 24 h. Suspend the cell pellet in 100 μl of FACS buffer and add 15.75 μl (20.75 μl when including CD271) of the human engraftment antibody cocktail.
In vivo HSC antibody cocktail
Prepare a master mix of the following antibody volumes for 1× samples: CD90 (2 μl), CD10 (5 μl), CD38 (1 μl), CD34 (2 μl), CD45RA (2.5 μl), CD45.1 (1 μl), and if using tNGFR as reporter gene, include CD271 (5 μl). Store at 4 °C in the dark for up to 24 h. Suspend the cell pellet in 100 μl and add 13.5 μl (18.5 μl when including CD271) of the in vivo HSC antibody master mix.
In vitro HSC antibody cocktail
Prepare a master mix of the following antibody volumes for 1× samples: CD34 (2 μl), CD45RA (2.5 μl), CD90 (2 μl), CD38 (1 μl), CD123 (5 μl), Lineage cocktail, 1 μl of each (CD2, CD3, CD4, CD8, CD16, CD19, CD20, CD56, CD235a (0.25 μl), and CD14), and if tNGFR is used as reporter gene, include CD271 (5 μl). Store at 4 °C in the dark for up to 24 h. Suspend the cell pellet in 100 μl and add 21.75 μl of in vitro HSC antibody master mix (26.75 μl if including CD271).
HSPC cytokine-rich medium
Culture all CD34+ HSPCs in Steriflip-filtered StemSpan SFEM II supplemented with SCF (100 ng/ml), TPO (100 ng/ml), Flt3L (100 ng/ml), IL-6 (100 ng/ml), UM171 (35 nM), 20 mg/ml streptomycin, 20 U/ml penicillin, and SR1 (StemRegenin1; 0.75 μM). Store at 4 °C for up to 1 week after preparation.
FACS buffer is 1× PBS supplemented with 2% (vol/vol) FBS and 2 mM EDTA. Store at 4 °C for up to 2 months.
Preculture of CD34+ HSPCs
Timing: 30 min–1 h hands-on; ∼2 d of culture
After HSPC isolation or thaw, seed the cells in HSPC cytokine-rich medium (Reagent Setup) at a density of 0.25–0.5E6 cells/ml and culture at 37 °C, 5% CO2, and 5% O2.
Culture the cells for 2 d (36–48 h). If the culture reaches 1 × 106 cells/ml, split to 0.25–0.5 × 106 cells/ml for optimal cell growth and viability, which also promotes high frequencies of HR.
Electroporation and transduction of cells
Timing: 1–2 h hands-on; 2–3 d of culture
On Day 2 after thaw/isolation, count the number of viable cells using a hemocytometer and Trypan blue dye exclusion51.
Prewarm an aliquot of HSPC cytokine-rich medium to 37 °C.
If using Lonza Nucleofection solutions, prepare solutions by mixing the entire supplement to the Nucleofector Solution (supplemented solution is active for 3 months). Alternatively, use the 1 M electroporation solution described in the Reagent Setup section. Allow the electroporation solution to equilibrate to room temperature.
Start the electroporation device and prepare the appropriate Nucleofector program. For the Lonza 4D device, we recommend the DZ100 program, and for the Lonza IIb, we recommend program U-014 for human CD34+ HSPCs.
Complex Cas9 protein with synthetic and chemically modified sgRNA (see the Supplementary Methods for identification, design, and production of sgRNA). Per 500,000–1,000,000 cells using nucleofection cuvettes: in a PCR tube, mix 15 μg of Cas9 protein with 8 μg of sgRNA (molar ratio of 1:2.5). For the Lonza 4D nucleocuvette strips, scale down fivefold. Mix well, centrifuge briefly (2,000g, 25 °C, 5 s), and complex by incubation in a thermocycler at 25 °C for 10 min. Store at 4 °C until use (up to 4 h) or on ice.
Meanwhile, pellet the desired number of cells (500,000–1,000,000 per electroporation) by centrifugation at room temperature, 300g for 5 min.
Remove the supernatant completely and resuspend the cell pellet in an appropriate volume of electroporation solution (100 μl per electroporation). Pipette up and down carefully to make a single-cell suspension.
Per electroporation, transfer 100 μl of cell suspension from Step 9 to the PCR tube with complexed Cas9 RNP from Step 7. Mix by pipetting gently and avoiding the formation of air bubbles. Transfer the entire volume to the electroporation cuvette and gently tap the cuvette to make sure that the sample covers the bottom of the cuvette.
Attach the lid to the cuvette, transfer it to the electroporation device, and electroporate the cells with the appropriate program selected in Step 6.
Directly after electroporation, add prewarmed HSPC cytokine-rich medium to the cuvette for a final cell density of 1 × 106 cells/ml. Mix very gently with a sterile transfer pipette and transfer the cells to a 24-well plate. Optionally, cells can be left to rest in the incubator for up to 20 min before transduction.
(Optional) Transfer 50,000 cells to a 96-well plate and adjust the cell density to 0.25 × 106 cells/ml using prewarmed HSPC cytokine-rich medium. This will serve as an 'RNP-only' sample to measure INDEL frequencies without the presence of the AAV6 donor.
Transduce the cells from Step 12 by adding AAV6 donor vector (prepared as described in the Supplementary Methods). Adjust the cell density with prewarmed HSPC cytokine-rich medium, so that the cells are transduced at a density of 0.25–0.5 × 106 cells/ml overnight and not for >24 h.
After overnight transduction or 24 h after transduction, add prewarmed HSPC cytokine-rich medium to the cells to reach a density of 0.25–0.5 × 106 cells/ml and transfer the cells to an appropriate cell culture dish.
Culture the cells for 2–3 d while not exceeding cell densities of 1 × 106 cells/ml. Depending on the transgene, promoter, and targeted locus, expression of the transgene can reach a maximum between Days 2 and 3 after electroporation and transduction.
Measurement of HR frequencies and confirmation of stem and progenitor phenotype
Timing: ∼1–2 h
Pellet at least 50,000 cells from each sample from Step 16 in two FACS tubes at room temperature, 300g for 5 min.
Aspirate the supernatant and resuspend in 100 μl of staining buffer.
Add antibody cocktail depending on which marker(s) to assess. Use option A to stain for HSPCs by CD34 expression or option B to stain for LT-HSCs and multipotent progenitors (MPPs).
Staining for HSPCs by CD34 expression
Add the CD34 antibody (and anti-NGFR antibody if used as reporter) to only one of the two tubes (the unstained CD34 tube will serve as negative control in flow cytometric analyses). Mix well and incubate on ice protected from light for 30 min. Optionally, include a sample stained with an isotype control antibody.
Staining for LT-HSCs and MPPs
It is possible to assess HR frequencies in CD34+ subpopulations: HSCs (Lin−/CD34+/CD38−/CD45RA−/CD90+), MPPs (Lin−/CD34+/CD38−/CD45RA−/CD90−), lymphoid-primed multipotent progenitors (LMPPs; Lin−, CD34+, CD38−, CD90−, and CD45RA+), common myeloid progenitors (CMPs; Lin−/CD34+/CD38+/CD45RA−/CD123+), granulocyte–macrophage progenitors (GMPs; Lin−/CD34+/CD38+/CD45RA+/CD123+), and megakaryocyte–erythrocyte progenitors (MEPs; Lin−/CD34+/CD38+/CD45RA−/CD123−). Representative FACS plots of subpopulations are depicted in Supplementary Figure 2 as well as in Figure 5f in Reinisch et al.52, Figure 1 in Majeti et al.27, and Figure 2a in Psaila et al.53. Add the in vitro HSC antibody cocktail, mix well, and incubate on ice in the dark for 30 min. Optionally, include samples stained with isotype control antibodies.
Fill the FACS tube with ice-cold staining buffer, pellet at 4 °C, 300g for 5 min, aspirate the supernatant, and resuspend in 200 μl of staining buffer with 1 μg/ml propidium iodide. Keep on ice in the dark until analysis.
Analyze on a flow cytometer, e.g., a FACS Aria (BD) using appropriate compensation controls and cytometer settings. Representative resulting FACS plots are shown in Figure 2 and Supplementary Figure 1. For the HSC antibody panel, use the gating strategy outlined in Supplementary Figure 2 or in Figure 1 in Park et al.46. Optionally, sort different populations of CD34+ cells, e.g., LT-HSCs and MPPs, from freshly purified CD34s and then subject to genome-editing to obtain HR frequencies in hematopoietic cells enriched for primitiveness (see Supplementary Figure 2 for representative FACS plots and gating scheme).
Performing CFU assay on targeted cells
Timing: 3–6 h hands-on; 12–16 d of culture
Thaw MethoCult medium at room temperature or overnight at 4 °C.
Shake the MethoCult medium vigorously for 1 min and let it stand for at least 5 min for bubbles to disappear.
With a 10-ml Luer lock syringe attached to a 16-gauge blunt-end needle, dispense one drop (∼100 μl) of MethoCult medium into each well of a flat-bottom 96-well plate, leaving all outer wells empty.
Fill the outer wells with either sterile PBS or water and put the plate into a 37 °C incubator.
Prepare a fluorescence-activated cell sorter (FACS, e.g., a FACS Aria) for aseptic single-cell sorting into 96-well plates.
Sort the targeted population of CD34+ HSPCs (from Step 16 as described in Steps 17–21) (reporterhigh population) one cell per well into the 60 wells with MethoCult medium from Step 24. In addition, sort the cells from a mock-electroporated sample, which will serve as a reference for total colony count and distribution of progenitor types.
Centrifuge the 96-well plate at room temperature, 300g for 5 min.
Incubate at 37 °C, 5% CO2, and 5% O2.
12–16 d after sorting, use a high-quality inverted microscope equipped to count colonies and score them according to morphology. Extensive guidelines on CFU scoring, as well as representative colony images can be found in the technical manual for MethoCult.
Extraction of genomic DNA from methylcellulose-derived colonies
Timing: 1–2 h
Circle the wells from Step 30 that contain colonies. Fill the wells with PBS and pipette up and down to dilute the viscous methylcellulose.
Transfer the contents of each circled well to a well of a U-bottom 96-well plate and pellet the cells at room temperature, 300g for 5 min.
Invert the plate on a tissue towel to remove the supernatant, resuspend the cells in 250 μl of PBS, and pellet at room temperature, 300g for 5 min.
Invert the plate on a tissue towel to remove the supernatant, add 25 μl of QuickExtract DNA Extraction Solution per well, and pipette up and down to resuspend the cells.
Transfer the cells to a PCR plate and incubate at 60 °C for 10 min, and then at 100 °C for 10 min.
PCR-based genotyping of methylcellulose-derived clones with targeted integration
Timing: 4–6 h
Design the primers as outlined in the Experimental design section and depicted in Figure 3.
To test if primers are functional and to find the optimal annealing temperature, use a thermocycler with gradient technology and pooled genomic DNA from at least ten clones as template DNA. Set up a PCR master mix for eight reactions (+1 extra):
Phusion PCR Master Mix (2×): 112.5 μl Primers (25 μM stock) 3 × 4.5 μl Template DNA 9 μl Nuclease-free water 90 μl Total volume 225 μl
Distribute the PCR master mix between eight PCR tubes (25 μl per tube).
Run the PCR reactions on a thermocycler programmed with the following thermal cycling parameters:
Cycle number Denature Anneal Extend Final 1 98 °C, 30 s 2–36 98°, 30 s Gradient from 57 to 72 °C, 30 s 72 °C, 30 s/kb 37 72 °C, 5 min 4 °C, ∞
Run each of the 25-μl PCR products and the GeneRuler 100 bp Plus DNA Ladder on a 1% (wt/vol) agarose gel at 100 V.
When the yellow dye present in the green PCR buffer has migrated to the bottom of the gel, visualize the gel on a UV gel-imaging system.
Identify the annealing temperature with the highest PCR efficiency and specificity.
For each of the colony-extracted genomic DNA samples from Step 35, repeat the PCR, following Steps 37–39 (scale the PCR master mix according to the total sample number) and using the optimal annealing temperature identified in Step 42, and run the PCR products on an agarose gel, following Steps 40–41.
Using a scalpel, excise the PCR fragments from the gel and gel-purify bands using the GeneJET Gel Extraction Kit and following the kit's instructions.
Sanger-sequence the purified PCR fragments using appropriate primers from the PCR reaction.
Align Sanger sequences with the reference sequence to validate on-target and seamless HR-mediated integration of the transgene cassette. Sanger sequencing of nonintegrated alleles may be performed for identification of INDELs. Note that it is possible (although infrequent) to obtain clones with incomplete HR events54. These frequencies must be empirically determined and may be noticeable by changes in PCR band sizes.
Transplantation of gene-modified CD34+ HSPCs
Timing: 3–10 h hands-on; 16 weeks of engraftment
24 h before transplantation, sublethally irradiate the desired number of 6- to 8-week-old NSG mice (preferably female47) with 200 cGy.
For transplantation of cells not enriched for targeting, skip to Step 53. Immediately before transplantation, phenotype cells from Step 16 for the presence of CD34, as described in Steps 17–21. For transplantation of enriched cells, prepare the cells as described in Steps 17–21 (prepare all the cells that are to be transplanted).
Prepare a FACS (e.g., a FACS Aria) for aseptic cell sorting into an appropriate-sized tube depending on the expected number of cells to be sorted.
Sort viable targeted cells (reporterhigh as depicted in Fig. 2). Optionally, include a CD34 stain and sort for CD34+ cells into prechilled PBS or HSPC cytokine-rich medium and collection tube holders chilled with 4 °C recirculating water.
Test the sort purity by analyzing a small subset of the sorted cells (250–500 cells) on the FACS. The purity should be >95%.
Using a hemocytometer and Trypan blue dye exclusion, count the number of sorted viable cells.
Pellet the desired number of cells at 4 °C, 300g for 5 min. Resuspend in 200 μl of PBS for intravenous transplantation or 25 μl of PBS for intrafemoral transplantation. Keep the cells on ice until transplantation is performed. Note that enriched targeted HSPCs have fewer absolute HSCs because of the reduced HR efficiencies in long-term repopulating HSCs; therefore, infusing a greater number of enriched targeted CD34+ HSPCs will usually lead to higher levels of engraftment. For tail-vein injection of sorted reporterhigh cells, we recommend transplanting at least 500,000 cells (cord blood–derived). For intrafemoral injections, we recommend transplantation of least 50,000 cells (cord blood–derived). Cells derived from mobilized peripheral blood or bone marrow usually require higher cell numbers to reach the same engraftment levels as those of cord blood–derived cells because of the lower fraction of LT-HSCs in the CD34+ population.
Transplant the cells either intrafemorally or intravenously (by tail-vein injection) using an insulin syringe with a 27-gauge × 1/2-inch needle into the recipient mice irradiated 24 h before transplantation.
Terminal analysis of engraftment in NSG mice at week 16
Timing: 5–10 h
Euthanize the mice according to the institutionally approved protocol.
Analyze engraftment in the bone marrow (option A), in which human chimerism is highest (five to ten times higher), or, if desired, analyze engraftment in the blood (option B) or spleen (option C).
Analysis of engraftment in the bone marrow
Dissect femurs, tibiae, humeri, pelvises, sternum, and vertebral column and remove the muscle and connective tissue. Transfer the bones to a mortar containing 5 ml of RPMI with 10% (vol/vol) FBS, 100 mg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 4 U/ml heparin, and 20 U/ml DNase.
Crush the bones using a pestle, and filter the liquid through a 100-μm filter into a 50-ml conical tube.
Add another 5 ml of RPMI containing 10% (vol/vol) FBS, 100 mg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 4 U/ml heparin, and 20 U/ml DNase to the mortar.
Repeat Step 56A(ii).
Incubate the cell suspension in a 37 °C water bath for 10 min for DNase treatment.
Add 40 ml of FACS buffer to the 50-ml conical tube. Using a pipette filler, fill a 10-ml serological pipette with 14 ml of Ficoll-Paque Plus and place the pipette at the bottom of the 50-ml conical tube. Remove the pipet filler and let gravity underlay the Ficoll-Paque Plus beneath the cell suspension. With a thumb against the end of the pipette, gently remove it from the 50-ml conical tube. Some Ficoll-Paque Plus will be retained in the pipette.
Centrifuge for 25 min at 850g at room temperature with full acceleration but no brake.
After centrifugation, aspirate the upper layer, leaving the mononuclear cell layer undisturbed.
Using a transfer pipette, carefully transfer the mononuclear cells to a new 50-ml conical tube.
Fill the 50-ml conical tube with ice-cold FACS buffer. Cells can be stored on ice for up to 4 h.
Analysis of engraftment in the blood
Using a 27-gauge insulin syringe, immediately after euthanasia, extract blood from the heart and transfer it to an Eppendorf tube containing 200 μl of PBS with 10 mM EDTA.
Add 500 μl of PBS with 2% (wt/vol) dextran to the blood sample and mix well.
Incubate at 37 °C for 30 min to sediment red blood cells. During incubation, the spleen can be processed following Step 56C(i–vii).
After 30-min incubation of the blood sample, transfer the leukocyte-rich plasma layer to a FACS tube while being careful to avoid disturbing the sedimented red blood cell layer.
Fill the FACS tube with ice-cold FACS buffer and centrifuge at 4 °C, 300g for 5 min to pellet the cells.
Aspirate the supernatant and resuspend the cells in 500 μl of red blood cell lysis buffer to lyse residual red blood cells. Incubate on ice for 5 min.
Fill the FACS tube with ice-cold FACS buffer and store on ice for up to 4 h.
Analysis of engraftment in the spleen
Dissect the spleen from the mouse and transfer to a six-well plate with 4 ml of RPMI containing 10% (vol/vol) FBS, 100 mg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 4 U/ml heparin, and 20 U/ml DNase.
Put the spleen on top of a 100-μm filter and put the filter against the RPMI solution in the same well. With the end of a syringe plunger, grind the spleen against the filter until it has disintegrated and the cells have passed through the filter mesh and into the RPMI solution.
Transfer the cells to a FACS tube through the 0.45-μm filter cap.
Incubate in a 37 °C water bath for 10 min for DNase treatment.
Fill the FACS tube with ice-cold FACS buffer and centrifuge at 4 °C, 300g for 5 min to pellet the cells.
Aspirate the supernatant and resuspend the cells in 500 μl of red blood cell lysis buffer to lyse residual splenic red blood cells. Incubate on ice for 5 min.
Fill the FACS tube with ice-cold FACS buffer and store on ice for up to 4 h.
Pellet the cells in a FACS tube by centrifugation at 4 °C, 300g for 5 min. Aspirate the supernatant, which should leave ∼100 μl of buffer in the tube.
Block Fc receptors by adding 5 μl of TruStain FcX, mix well, and incubate at room temperature for 5 min.
Add the human engraftment antibody cocktail, mix well, and incubate on ice in the dark for 30 min. Optionally, if the flow cytometer allows, the antibody panel can be expanded to stain for markers of hematopoietic lineages other than B cells and myeloid cells.
Fill the FACS tube with ice-cold FACS buffer and pellet the cells by centrifugation at 4 °C, 300g for 5 min.
Aspirate the supernatant and resuspend in 400 μl of ice-cold FACS buffer with 1 μg/ml propidium iodide.
Analyze on a flow cytometer, e.g., a FACS Aria with proper compensation settings and controls.
(Optional) If desired, analyze the presence of engrafted CD34+ human cells in the bone marrow of the primary recipients at week 16. Stain 5 ml of the cells from Step 56A(x) for HSPC markers (CD34+, CD38−, and CD10−) using the in vivo HSC antibody cocktail and following Steps 57–62. Note that CD10 negatively discriminates for immature B cells that are also CD34+.
(Optional) Establish single-cell-derived methylcellulose clones from CD34+ human cells in the bone marrow of the primary recipients at week 16. This enables precise clonal genotyping of the long-term repopulating cells. Single-cell sort stained cells from Step 63 (CD34+, CD38−, and CD10−) into 96-well plates with methylcellulose, as outlined in Steps 22–30. Extract gDNA as described in Steps 31–35 and perform PCR-based genotyping described in Steps 36–46.
(Optional) Perform secondary transplantations to assess true long-term hematopoietic reconstitution potential using the remaining bone marrow-derived mononuclear cells not stained for human engraftment in Step 56A(x). Irradiate NSG mice 24 h before (Step 47) and transplant according to Steps 48–54. Before pelleting and resuspension in Step 53, filter the cells through a 45-μm mesh. Alternatively, for low-engrafting mice, it is advised to pool MNCs and enrich for CD34+ cells using the MACS CD34 MicroBead Kit. We recommend transplanting 5–10 million mononuclear cells intravenously or 2 million mononuclear cells intrafemorally. Human engraftment in secondary mice should be analyzed at least 12 weeks after transplantation, following Steps 55–62 (Fig. 5).
Troubleshooting advice can be found in Table 1.
Steps 1 and 2, isolation/thawing and preculture of CD34+ cells: 30 min–1 h hands-on, 2 d of culture
Steps 3–16, electroporation and transduction of cells: 1–2 h hands-on, 2 to 3 d of culture
Steps 17–21, measurement of HR frequencies and confirmation of stem and progenitor phenotype: 1–2 h hands-on
Steps 22–30, performing of CFU assay on targeted cells: 3–6 h hands-on, 12 to 16 d of culture
Steps 31–35, extraction of genomic DNA from methylcellulose-derived colonies: 1–2 h hands-on
Steps 36–46, genotyping of clones for targeted integration using PCR: 4–6 h hands-on
Steps 47–54, transplantation of gene-modified CD34+ cells: 3–10 h hands-on, 16 weeks of engraftment
Steps 55–65, performing terminal analysis of engraftment in NSG mice: 5–10 h hands-on
Box 1, homologous recombination in primary human T cells: 6–8 h hands-on, 5–7 d of culture
Genome editing by HR in HSCs allows for the investigation of HSC gene function, as well as for the ex vivo correction of disease-causing mutations for the development of the next generation of gene and cell therapies. By combining the CRISPR/Cas9 system delivered as RNPs with homologous donor delivery via rAAV6, we routinely achieve >20% (and up to 77%) HR frequencies in CD34+ HSPCs in vitro at several human loci, and assess reporterhigh expression via FACS after successful HR into the intended locus 1–3 d post electroporation. Figure 2 and Supplementary Figure 1 display the expected results from a successful HR experiment with a reporterhigh population present only when the CRISPR components are added to the cells. Importantly, sorting of this reporterhigh HSPC population enriches for cells with targeted integration, as shown in Figure 2, and also circumvents potential inefficient HSC targeting by removing untargeted HSCs that can compete for reconstitution in the bone marrow. CFU assays generally result in 30–50% colony formation with >60% of colonies being CFU-GM and <5% being CFU-GEMM, but the results can vary depending on HSPC source. Upon transplantation into NSG mice, we generally observe human engraftment in all transplanted mice, and with transplantation of enriched HSPCs, we generally observe that >80% of the human cells are reporter+. Figure 4 shows the expected results after flow cytometric analysis of bone marrow engrafted with transplanted human cells. We envision that this methodology will be widely used for studying HSPC biology and advancing the field of gene and cell therapy for blood and immune system disorders.
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R.O.B. was supported through an individual postdoctoral grant (DFF-1333-00106B) and a Sapere Aude, Research Talent grant (DFF-1331-00735B), both from the Danish Council for Independent Research, Medical Sciences. D.P.D. was supported through a Stanford Child Health Research Institute (CHRI) grant and postdoctoral award. M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research, and NIH grants R01-AI097320 and R01-AI120766. We further thank members of the Porteus and Majeti lab for helpful input, comments, and discussions.
Integrated supplementary information
Supplementary Figures 1–3 and the Supplementary Methods.