{"id":615989,"date":"2024-06-16T20:00:00","date_gmt":"2024-06-17T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/enhancement-of-erythropoietic-output-by-cas9-mediated-insertion-of-a-natural-variant-in-haematopoietic-stem-and-progenitor-cells-nature-biomedical-engineering\/"},"modified":"2024-06-17T12:23:33","modified_gmt":"2024-06-17T16:23:33","slug":"enhancement-of-erythropoietic-output-by-cas9-mediated-insertion-of-a-natural-variant-in-haematopoietic-stem-and-progenitor-cells-nature-biomedical-engineering","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/enhancement-of-erythropoietic-output-by-cas9-mediated-insertion-of-a-natural-variant-in-haematopoietic-stem-and-progenitor-cells-nature-biomedical-engineering\/","title":{"rendered":"Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells – Nature Biomedical Engineering","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
<\/div>\n

AAV6 vector design, production and purification<\/h3>\n

AAV6 vector plasmids were cloned into the pAAV-MCS plasmid (Agilent Technologies) comprising inverted terminal repeats derived from AAV2. Gibson Assembly Master Mix (New England Biolabs) was used for the creation of all DNA repair vectors as per the manufacturer\u2019s instructions. AAV6 vector was produced and purified with little variation from previously described processes41<\/a><\/sup>. 293T cells (Life Technologies) were seeded in five dishes (15 cm2<\/sup>) with 13\u2009\u00d7\u2009106<\/sup>\u201315\u2009\u00d7\u2009106<\/sup> cells per plate at 24-h pretransfection. Each dish was then transfected with a standard polyethylenimine (PEI) transfection of 6\u2009\u03bcg inverted-terminal-repeat-containing plasmid and 22\u2009\u03bcg pDGM6 (gift from David Russell, University of Washington), which holds the AAV6 cap<\/i>, AAV2 rep<\/i> and Ad5 helper<\/i> genes. After a 48\u201372-h incubation, cells were collected and vectors were purified using the AAVpro purification kit (catalogue number 6666; Takara Bio) per the manufacturer\u2019s instructions and then stored at \u221280\u2009\u00b0C until further use. AAV6 vectors were titred using ddPCR to measure the number of vector genomes as previously described42<\/a><\/sup>.<\/p>\n

In vitro culture of CD34+<\/sup> HSPCs<\/h3>\n

Human CD34+<\/sup> HSPCs were cultured in conditions as previously described13<\/a>,43<\/a>,44<\/a>,45<\/a>,46<\/a><\/sup>. CD34+<\/sup> HSPCs were isolated from cord blood (provided by Stanford Binns Program for Cord Blood Research) or sourced from plerixafor- and\/or G-CSF-mobilized peripheral blood (AllCells and STEMCELL Technologies). Frozen plerixafor- and\/or G-CSF-mobilized peripheral blood of patients with SCD were provided by Dr Vivien Sheehan at Emory University. CD34+<\/sup> HSPCs were cultured at 1\u2009\u00d7\u2009105<\/sup>\u20135\u2009\u00d7\u2009105<\/sup> cells\u2009ml\u22121<\/sup> in StemSpan Serum-Free Expansion Medium II (STEMCELL Technologies) or Good Manufacturing Practice Stem Cell Growth Medium (SCGM; CellGenix) supplemented with a human cytokine (PeproTech) cocktail: stem cell factor (100\u2009ng\u2009ml\u22121<\/sup>), thrombopoietin (100\u2009ng\u2009ml\u22121<\/sup>), Fms-like tyrosine kinase 3 ligand (100\u2009ng\u2009ml\u22121<\/sup>), interleukin-6 (100\u2009ng\u2009ml\u22121<\/sup>), streptomycin (20\u2009mg\u2009ml\u22121<\/sup>), penicillin (20\u2009U\u2009ml\u22121<\/sup>) and 35\u2009nM of UM171 (catalogue number A89505; APExBIO). The cell incubator conditions were 37\u2009\u00b0C, 5% CO2<\/sub> and 5% O2<\/sub>.<\/p>\n

Electroporation-aided transduction of cells<\/h3>\n

The synthetic chemically modified sgRNAs used to edit CD34+<\/sup> HSPCs were purchased from Synthego or TriLink Biotechnologies and were purified by HPLC. These modifications comprise 2\u2032-O<\/i>-methyl-3\u2032-phosphorothioate at the three terminal nucleotides of the 5\u2032 and 3\u2032 ends described previously17<\/a><\/sup>. The target sequences for the gRNAs were as follows.<\/p>\n

\n EPOR<\/i> gRNA (EPOR<\/i>-sg1)<\/h4>\n

5\u2032-AGCTCAGGGCACAGTGTCCA-3\u2032<\/p>\n

\n EPOR<\/i> gRNA (EPOR<\/i>-sg2)<\/h4>\n

5\u2032-GCTCCCAGCTCTTGCGTCCA-3\u2032<\/p>\n

\n CCR5<\/i> gRNA (CCR5-<\/i>sg3)<\/h4>\n

5\u2032-GCAGCATAGTGAGCCCAGAA-3\u2032<\/p>\n

\n HBA1<\/i> gRNA (HBA1<\/i>-sg4)<\/h4>\n

5\u2032-GGCAAGAAGCATGGCCACCG-3\u2032<\/p>\n

The HiFi Cas9 protein was purchased from Integrated DNA Technologies (IDT) or Aldevron. Before electroporation, RNPs were complexed at a Cas9\/sgRNA molar ratio of 1:2.5 at 25\u2009\u00b0C for 10\u201320\u2009min. Next, CD34+<\/sup> cells were resuspended in P3 buffer (Lonza) with complexed RNPs and subsequently electroporated using the Lonza 4D-Nucleofector and 4D-Nucleofector X Unit (program DZ-100). Electroporated cells were then plated at 1\u2009\u00d7\u2009105<\/sup>\u20135\u2009\u00d7\u2009105<\/sup> cells\u2009ml\u22121<\/sup> in the previously described cytokine-supplemented media. Immediately after electroporation, AAV6 was dispensed onto cells at 2.5\u2009\u00d7\u2009103<\/sup>\u20135\u2009\u00d7\u2009103<\/sup> vector genomes per cell based on titre determined by ddPCR. For multiplex editing experiments, in addition to the steps described above, cells were incubated with 0.5\u2009\u03bcM of the DNA-PKcs inhibitor AZD7648 (catalogue number S8843; Selleck Chemicals) for 24\u2009h, as previously described32<\/a>,33<\/a><\/sup>.<\/p>\n

Allelic modification analysis using ddPCR<\/h3>\n

Edited HSPCs were collected within 2\u20133 days postelectroporation and at each media change throughout erythrocyte differentiation and then analysed for modification frequencies of the alleles of interest. To quantify editing frequencies, we created custom ddPCR primers and probes to quantify HDR alleles (using in\u2013out PCR and probe corresponding to the expected integration event) compared with an established genomic DNA reference (REF) at the CCRL2<\/i> locus14<\/a><\/sup>. QuickExtract DNA extraction solution (catalogue number QE09050; Biosearch Technologies) was used to collect genomic DNA input, which was then digested using BamHI-HF or HindIII-HF as per the manufacturer\u2019s instructions (New England Biolabs). The percentage of targeted alleles within a cell population was measured with a Bio-Rad QX200 ddPCR machine and QuantaSoft software (v.1.7; Bio-Rad) using the following reaction mixture: 1\u20134\u2009\u03bcl genomic DNA input, 10\u2009\u03bcl of ddPCR Supermix for Probes (no dUTP; Bio-Rad), primer and probes (1:3.6 ratio; IDT), and volume up to 20\u2009\u03bcl with H2<\/sub>O. ddPCR droplets were then generated following the manufacturer\u2019s instructions (Bio-Rad): 20\u2009\u03bcl of ddPCR reaction, 70\u2009\u03bcl of droplet generation oil and 40\u2009\u03bcl of droplet sample. Thermocycler (Bio-Rad) settings were as follows: 98\u2009\u00b0C (10\u2009min), 94\u2009\u00b0C (30\u2009s), 55.7\u201360\u2009\u00b0C (30\u2009s), 72\u2009\u00b0C (2\u2009min), return to step 2 for 40\u201350 cycles and then 98\u2009\u00b0C (10\u2009min). Analysis of droplet samples was then performed using the QX200 Droplet Digital PCR System (Bio-Rad). We next divided the copies per microlitre for HDR (%): HDR\/REF. The following primers and probes were used in the ddPCR reaction.<\/p>\n

\n CCR5<\/i> (for tEPOR<\/i>–YFP<\/i> construct)<\/h4>\n

Forward primer (FP): 5\u2032-GGGAGGATTGGGAAGACA-3\u2032<\/p>\n

Reverse primer (RP): 5\u2032-AGGTGTTCAGGAGAAGGACA-3\u2032<\/p>\n

Probe: 5\u2032-6-FAM\/AGCAGGCATGCTGGGGATGCGGTGG\/3IABkFQ-3\u2032<\/p>\n

\n HBA1<\/i> (for tEPOR<\/i>–YFP<\/i> construct)<\/h4>\n

FP: 5\u2032-AGTCCAAGCTGAGCAAAGA-3\u2032<\/p>\n

RP: 5\u2032-ATCACAAACGCAGGCAGAG-3\u2032<\/p>\n

Probe: 5\u2032-6-FAM\/CGAGAAGCGCGATCACATGGTCCTGC\/3IABkFQ-3\u2032<\/p>\n

\n HBA1<\/i> (for HBB<\/i> construct and tEPOR-HBB<\/i> constructs)<\/h4>\n

FP: 5\u2032-GTGGCTGGTGTGGCTAATG-3\u2032<\/p>\n

RP: 5\u2032-CAGAAAGCCAGCCAGTTCTT-3\u2032<\/p>\n

Probe: 5\u2032-6-FAM\/CCTGGCCCACAAGTATCACT\/3IABkFQ-3\u2032<\/p>\n

\n HBA1<\/i> (for HBB-tEPOR<\/i> constructs)<\/h4>\n

FP: 5\u2032-TCTGCTGCCAGCTTTGAGTA-3\u2032<\/p>\n

RP: 5\u2032-GCTGGAGTGGGACTTCTCTG-3\u2032<\/p>\n

Probe: 5\u2032-6-FAM\/ACTATCCTGGACCCCAGCTC\/3IABkFQ-3\u2032<\/p>\n

\n CCRL2<\/i> (reference)<\/h4>\n

FP: 5\u2032-GCTGTATGAATCCAGGTCC-3\u2032<\/p>\n

RP: 5\u2032-CCTCCTGGCTGAGAAAAAG-3\u2032<\/p>\n

Probe: 5\u2032-HEX\/TGTTTCCTC\/ZEN\/CAGGATAAGGCAGCTGT\/3IABkFQ-3\u2032<\/p>\n

Indel analysis using TIDE software<\/h3>\n

Within 2\u20134 days postelectroporation, HSPCs were collected with QuickExtract DNA extraction solution (catalogue number QE09050; Biosearch Technologies) to collect genomic DNA. The following primer sequences were used to amplify the respective cut sites at the EPOR<\/i> locus:<\/p>\n

FP: 5\u2032-CAGCTGTGGCTGTACCAGAA-3\u2032<\/p>\n

RP: 5\u2032-CAGCCTGGTGTCCTAAGAGC-3\u2032<\/p>\n

Sanger sequencing of the respective samples was then used as input for indel frequency analysis relative to a mock, unedited sample using TIDE as previously described25<\/a><\/sup>.<\/p>\n

In vitro differentiation of CD34+<\/sup> HSPCs into erythrocytes<\/h3>\n

Following editing, HSPCs derived from healthy individuals or patients with SCD were cultured for 2\u20133 days as described above. Subsequently, a 14-day in vitro differentiation was performed in supplemented SFEMII medium as previously described24<\/a>,47<\/a><\/sup>. SFEMII base medium was supplemented with 100\u2009U\u2009ml\u22121<\/sup> penicillin\u2013streptomycin, 10\u2009ng\u2009ml\u22121<\/sup> SCF (PeproTech), 1\u2009ng\u2009ml\u22121<\/sup> IL-3 (PeproTech), 3\u2009U\u2009ml\u22121<\/sup> EPO (eBiosciences), 200\u2009\u03bcg\u2009ml\u22121<\/sup> transferrin (Sigma-Aldrich), 3% human serum (heat-inactivated; Sigma-Aldrich or Thermo Fisher Scientific), 2% human plasma (isolated from umbilical cord blood provided by the Stanford Binns Cord Blood Program), 10\u2009\u03bcg\u2009ml\u22121<\/sup> insulin (Sigma-Aldrich) and 3\u2009U\u2009ml\u22121<\/sup> heparin (Sigma-Aldrich). Cells were cultured in the first phase of medium for 7 days at 1\u2009\u00d7\u2009105<\/sup> cells\u2009ml\u22121<\/sup>. In the second phase of medium, days 7\u201310, cells were maintained at 1\u2009\u00d7\u2009105<\/sup> cells\u2009ml\u22121<\/sup> and IL-3 was removed from the culture. In the third phase of medium, days 11\u201314, cells were cultured at 1\u2009\u00d7\u2009106<\/sup> cells\u2009ml\u22121<\/sup>, with a transferrin increase to 1\u2009mg\u2009ml\u22121<\/sup>.<\/p>\n

Immunophenotyping of differentiated erythrocytes<\/h3>\n

Differentiated erythrocytes were analysed by flow cytometry on day 14 for erythrocyte lineage-specific markers using a FACS Aria II (BD Biosciences). Edited and unedited cells were analysed using the following antibodies: hCD45-V450 (HI30; BD Biosciences), CD34-APC (561; BioLegend), CD71-PE-Cy7 (OKT9; Affymetrix) and CD235a-PE (GPA) (GA-R2; BD Biosciences). In addition to cell-specific markers, cells were also stained with Ghost Dye Red 780 (Tonbo Biosciences) to measure viability.<\/p>\n

Haemoglobin tetramer analysis<\/h3>\n

Frozen pellets of approximately 1\u2009\u00d7\u2009106<\/sup> in vitro-differentiated erythrocytes were thawed and lysed in 30\u2009\u00b5l of RIPA buffer with 1\u00d7 Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) for 5\u2009min on ice. The mixture was vigorously vortexed and cell debris was removed by centrifugation at 13,000\u2009r.p.m. for 10\u2009min at 4\u2009\u00b0C. HPLC analysis of haemoglobins in their native form was performed on a cation-exchange PolyCAT A column (35\u2009mm2<\/sup>\u2009\u00d7\u20094.6\u2009mm2<\/sup>, 3\u2009\u00b5m, 1,500\u2009\u00c5; PolyLC) using a Perkin-Elmer Flexar HPLC system at room temperature and detection at 415\u2009nm. Mobile phase A consisted of 20\u2009mM Bis-Tris and 2\u2009mM KCN at pH 6.94, adjusted with HCl. Mobile phase B consisted of 20\u2009mM Bis-Tris, 2\u2009mM KCN and 200\u2009mM NaCl at pH 6.55. Haemolysate was diluted in buffer A before injection of 20\u2009\u00b5l onto the column with 8% buffer B and eluted at a flow rate of 2\u2009ml\u2009min\u22121<\/sup> with a gradient made to 40% B in 6\u2009min, increased to 100% B in 1.5\u2009min, returned to 8% B in 1\u2009min and equilibrated for 3.5\u2009min. Quantification of the area under the curve of the peaks was performed with TotalChrom software (Perkin-Elmer) and raw values were exported to GraphPad Prism 9 for plotting and further analysis.<\/p>\n

mRNA analysis<\/h3>\n

After differentiation of HSPCs into erythrocytes, cells were collected and RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Subsequently, cDNA was made from approximately 100\u2009ng of RNA using the iScript Reverse Transcription Supermix for quantitative PCR with reverse transcription (Bio-Rad). Expression levels of the \u03b2-globin<\/i> transgene and \u03b1-globin<\/i> mRNA were quantified with a Bio-Rad QX200 ddPCR machine and QuantaSoft software (v.1.7; Bio-Rad) using the following primers and 6-FAM\/ZEN\/IBFQ-labelled hydrolysis probes, purchased as custom-designed PrimeTime qPCR Assays from IDT.<\/p>\n

\n HBB<\/i> and HBB-tEPOR<\/i> into HBA1<\/i>
\n <\/h4>\n

FP: 5\u2032-GGTCCCCACAGACTCAGAGA-3\u2032<\/p>\n

RP: 5\u2032-CAGCATCAGGAGTGGACAGA-3\u2032<\/p>\n

Probe: 5\u2032-6-FAM\/AACCCACCATGGTGCATCTG\/3IABkFQ-3\u2032<\/p>\n

To normalize for RNA input, levels of the RBC-specific reference gene GPA<\/i> were determined in each sample using the following primers and HEX\/ZEN\/IBFQ-labelled hydrolysis probes, purchased as custom-designed PrimeTime qPCR Assays from IDT.<\/p>\n

\n GPA<\/i> (reference)<\/h4>\n

FP: 5\u2032-ATATGCAGCCACTCCTAGAGCTC-3\u2032<\/p>\n

RP: 5\u2032-CTGGTTCAGAGAAATGATGGGCA-3\u2032<\/p>\n

Probe: 5\u2032-HEX\/AGGAAACCGGAGAAAGGGTA\/3IABkFQ-3\u2032<\/p>\n

ddPCR reactions were created using the respective primers and probes and droplets were generated as described above. Thermocycler (Bio-Rad) settings were as follows: 98\u2009\u00b0C (10\u2009min), 94\u2009\u00b0C (30\u2009s), 54\u2009\u00b0C (30\u2009s), 72\u2009\u00b0C (30\u2009s), return to step 2 for 50 cycles and then 98\u2009\u00b0C (10\u2009min). Analysis of droplet samples was done using the QX200 Droplet Digital PCR System (Bio-Rad). To determine relative expression levels, the numbers of HBB<\/i> transgene copies per millilitre were divided by the numbers of GPA<\/i> copies\u2009ml\u22121<\/sup>.<\/p>\n

Methylcellulose CFU assay<\/h3>\n

At 2\u20133 days postelectroporation, HSPCs were plated in SmartDish 6 well plates (catalogue number 27370; STEMCELL Technologies) containing MethoCult H4434 Classic or MethoCult H4434 Classic without EPO (catalogue numbers 04444 and 04544; STEMCELL Technologies). After 14 days, the wells were imaged using the STEMvision Hematopoietic Colony Counter (STEMCELL Technologies). Colonies were counted and scored to determine the number of CFU-GEMM (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte), CFU-GM (colony-forming unit-granulocyte, macrophage), BFU-E (burst-forming unit-erythroid) and CFU-E (colony-forming unit-erythroid) colonies.<\/p>\n

Quantification of editing efficiency at evaluated off-target sites<\/h3>\n

Potential sgRNA off-target sites were predicted using the CRISPR Off-target Sites with Mismatches, Insertions and Deletions (COSMID) online tool48<\/a><\/sup>. Sites were ranked according to score and duplicate predictions at the same location were removed. All sites with a score \u22645.5 were included in the analysis and the 5 sites in exonic or untranslated regions were further analysed. PCR amplification of these sites was performed using genomic DNA from mock-edited and RNP-edited cells. The following primers were used with Illumina adaptors (FP adaptor, 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3\u2032; RP adaptor, 5\u2032-GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3\u2032).<\/p>\n

\n EPOR-OT1<\/i>
\n <\/h4>\n

FP: 5\u2032-GAGCGGGCTACAGAGCTAGA-3\u2032<\/p>\n

RP: 5\u2032-TGGCAGAAAGTAAGGGGATG-3\u2032<\/p>\n

\n EPOR-OT2<\/i>
\n <\/h4>\n

FP: 5\u2032-ACTTGTGGAGCCACAGTTTG-3\u2032<\/p>\n

RP: 5\u2032-AATGCCCTTGAGATGAATGC-3\u2032<\/p>\n

\n EPOR-OT3<\/i>
\n <\/h4>\n

FP: 5\u2032-TCACACACCCGTAGCCATAA-3\u2032<\/p>\n

RP: 5\u2032-AGAATGCTCTTTGCAGTAGCC-3\u2032<\/p>\n

\n EPOR-OT4<\/i>
\n <\/h4>\n

FP: 5\u2032-CTCAAAACTTCACCCAGGCT-3\u2032<\/p>\n

RP: 5\u2032-GGTCTGTCATTGAATGCCTT-3\u2032<\/p>\n

\n EPOR-OT5<\/i>
\n <\/h4>\n

FP: 5\u2032-CAACCCTGATGGGTCTGC-3\u2032<\/p>\n

RP: 5\u2032-CCACAGCTGGCTGACCTT-3\u2032<\/p>\n

Following amplification, PCR products were purified by gel electrophoresis and subsequent extraction using the GeneJet Gel Extraction Kit (catalogue number FERK0692; Thermo Fisher Scientific). Purified samples were submitted for library preparation and sequencing by Amplicon-EZ next-generation sequencing (Azenta Life Sciences), ensuring a yield of over 100,000 reads per sample. Amplicons, flanked by Illumina partial adaptor sequences, which encompassed the programmed DSBs for CRISPR\u2013Cas9, underwent sequencing using Illumina chemistry. FastQC (v.0.11.8, default parameters; http:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/<\/a>) was used to assess the quality of raw reads. Subsequently, paired-end reads were aligned to the specified off-target regions using CRISPResso2 (v.2.2.14; fastq.gz files were used as input)49<\/a><\/sup>.<\/p>\n

Statistical analysis<\/h3>\n

GraphPad Prism 9 software was used for all statistical analysis.<\/p>\n

Reporting summary<\/h3>\n

Further information on research design is available in the Nature Portfolio Reporting Summary<\/a> linked to this article.<\/p>\n