2017
DOI: 10.1186/s13045-017-0489-9
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Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing

Abstract: BackgroundSickle cell disease (SCD) is a disorder of red blood cells (RBCs) expressing abnormal hemoglobin-S (HbS) due to genetic inheritance of homologous HbS gene. However, people with the sickle cell trait (SCT) carry a single allele of HbS and do not usually suffer from SCD symptoms, thus providing a rationale to treat SCD.MethodsTo validate gene therapy potential, hematopoietic stem cells were isolated from the SCD patient blood and treated with CRISPR/Cas9 approach. To precisely dissect genome-editing ef… Show more

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Cited by 26 publications
(27 citation statements)
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“…Recent reports suggest that the RNP catalyzes high levels of gene editing activity at the beta globin locus [7,8,18] and in some cases, executes HDR with minimal indel formation [19]. The schematic in Fig.…”
Section: Schematic Of Crispr Ribonucleoprotein and Overall Workflow Omentioning
confidence: 99%
See 1 more Smart Citation
“…Recent reports suggest that the RNP catalyzes high levels of gene editing activity at the beta globin locus [7,8,18] and in some cases, executes HDR with minimal indel formation [19]. The schematic in Fig.…”
Section: Schematic Of Crispr Ribonucleoprotein and Overall Workflow Omentioning
confidence: 99%
“…Investigators have begun to coalesce around an RNP particle consisting of a specific guide (sg)RNA complexed with a bacterial nuclease, most often Cas9 [4][5][6] as the preferred editing tool. It is now known that the RNP can be effectively delivered to progenitor cells ex vivo and in combination with a single-stranded donor DNA template, can execute single nucleotide correction [7,8]. The singlestranded DNA donor can be generated by replication and expression of single-stranded DNA from Adeno-associated virus template [9] or synthetic single-stranded DNA oligonucleotides (ssODNs) [10][11][12].…”
Section: Introductionmentioning
confidence: 99%
“… Mobilized peripheral blood NA (X-VIV015 media + cytokine supplements) 500,000 P3 Primary Cell 4D-Nucleofector kit Lonza 4D-Nucleofector (program EO-100) 10 μg sgRNA, 15 μg Cas9 mRNA. and 1 μg plasmid 28 Mobilized peripheral BM and CB 1–2 days cells in culture before transfection (StemSpan SFEM II media + listed cytokines) 5 million–80 million Human T Cell Nucleofector kit Nucleofector 2b device (program U-014) Cas9 protein/sgRNA or Cas9 mRNA/sgRNA + AAV6 donor vector 2 Mobilized peripheral blood 24 hr cultured cells before transfection (StemSpan SFEM + CC110 cocktail) 100,000–200,000 Lorita P3 solution Lonza 4D-Nucleofector (program 00100 or ER1 OO) 72–200 pmol Cas9 RNP + 100 μM ssDNA 26 Peripheral blood of SC patients cells expanded 7 days before transfection (StemSpan SFEM II + CC100 cytokine cocktail) 5,000–10,000 Human CD34 Cell Nucleofector kit Nucleofector 2b device (program U-008) 1st electroporation, l0μg Cas9 mRNA; 2nd electroporation, sgRNA and HDR template 29 Peripheral blood and CB 2 days in culture before transfection (StemSpan + cytokines) 150,000–250,000 Neon Transfection System (10 μL kit) Neon Transfection System (1,600 V, 10 ms, 3 pulses) 200 ng-1 μg sgRNAs + 1 μg Cas9 protein 6 CB and BM of SCD patients 2 days in culture before transfection (X-VIV015 + cytokines) 200,000 BTXpress buffer BTX ECM 830 Square Wave Electroporator (250 V, 5 ms) ZFN mRNA + oligonucleotide 30 Mobilized peripheral blood NA (not easily found) 1 million–5 million NA Nucleofector 2b device (program U-008) 2 × 10 μg DNA plasmid encoding gRNA, Cas9, and GFP 31 The options available for the delivery of gene editing tools into CD34+ cells. BM, bone marrow; CB, cord blood; HDR, homology-directed repair; NA, not applicable; ssDNA, single-stranded DNA; SC, sickle cell; ZFN, zinc-finger nuclease.…”
Section: Discussionmentioning
confidence: 99%
“…The gene correction strategy for β‐hemoglobinopathies using the CRISPR system was tested in different human cell sources including patient/donor‐derived induced pluripotent stem cells (iPSCs), HSCs and human embryos (Table ). For this approach, studies were mostly targeting the sites near the SCD mutation ( HBB :c.20A>T, p.E6V; rs334) and most common β‐thalassemia mutation, that is, 4‐bp deletion at codon 41/42 ( HBB :c.124_127delTTCT/ HBB :c.126_129delCTTT) which is prevalent in East Asia and Southeast Asia . Moreover, a study had tried to correct a common β‐thalassemia splicing variant IVS2‐654 ( HBB :c.316‐197C>T) .…”
Section: Crispr/cas9 In Inherited Nonmalignant Hematological Blood DImentioning
confidence: 99%