1c and Additional file 1: Table S4). Just over 49,000 times! 2013;288(48):34671–9.

Google Scholar.

Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems.

Of these seven gRNAs, gRNA1, which targeted DSB at two nucleotides upstream of the HbE point mutation, gave the highest efficiency and was then selected for further experiments (Fig. Several iPSC lines, designated Eβ-iPSCs, were obtained. CAS  We performed an efficient one-step seamless genetic correction of iPSCs from a HbE/β-thalassemic patient using the CRISPR/Cas9 system and the ssODN template. We obtained human dermal fibroblasts (HDFs) from a patient with HbE/β-thalassemia who had a 4-bp deletion (–TCTT) at codon 41/42 in one allele, and a point mutation (G → A) at codon 26 resulting in abnormal hemoglobin E (HbE) production in the other, and generated iPSCs from these HbE/β-thalassemia patient’s HDFs. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. Your best ratio will depend on what type of PEI, linear or branched, what molecular weight etc. A previous study demonstrated that the transcription factors KLF1 and BCL11A are required for the induction of beta hemoglobin levels [38]; therefore, low levels of KLF1 and BCL11A transcripts in the corrected C46 cell-derived erythroid cells could result in lower beta hemoglobin expression. From a total of 312 individual clones screened, we estimated that 93 clones were transfected (according to 30% transfection efficiency with the GFP plasmid control). Red arrowheads show mutation sites; black arrowhead shows cleavage site of Cas9 by gRNA1; purple and red arrows indicate primer pairs for T7E1 assay and multiplex PCR for HbE detection, respectively. All patients were provided with an explanation and with a participant information sheet and signed the informed consent. Correspondence to

Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. Xu P, Tong Y, Liu X-Z, Wang T-T, Cheng L, Wang B-Y, et al.

We have clonned our gRna into pSpCas9(BB)-2A-Puro (PX459) V2.0. but a GFP signal is only visible in the cell also expressing cav1-GFP11.

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. (AVI).

Lengerke C, Daley GQ. The protein concentration was measured by the Pierce™ BCA Protein Assay Kit (Thermo Scientific). To improve HDR efficiency, small molecule inhibitors may be used to suppress the nonhomologous end joining (NHEJ) pathway, thus facilitating the downstream screening process [27, 28]. Naïve induced pluripotent stem cells generated from β-thalassemia fibroblasts allow efficient gene correction with CRISPR/Cas9. HbE mutation was detected by multiplex PCR. We’re very excited for more reagent sharing projects in the years to come. Recently, an alternative CRISPR system to Cas9 nuclease called Cpf1 nuclease was used for genome editing with simpler crRNA synthesis and more effective delivery and targeting results [37]. I have obtained pSpCas9(BB)-2A-Puro vector from Dr. Zhang's lab via Addgene. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. 2c, d). J Biol Chem. The latter can be achieved via nickases that induce single-strand breaks [32, 33] or single-guide RNA (sgRNA)-guided catalytically inactive Cas9 (dCas9) fused to the FokI nuclease [34,35,36]. Manage cookies/Do not sell my data we use in the preference centre. Inside Addgene, TRG5880200 to MW) and the Commission on Higher Education (grant no. Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C > T) mutation in β-thalassemia-derived iPSCs. Adherent cells were dissociated with accutase at 37 °C for 15 min, washed twice with PBS/EDTA + 2% FBS and stained with antibodies specific to hematopoietic markers (CD34-PE, CD43-FITC, CD235a-PE and CD71-FITC; all from BioLegend) at room temperature for 15 min.

However, two of the corrected clones (C134 and C258) could only give rise to CFU-GM and a small number of CFU-E (Fig. The clinical manifestations are heterogeneous; at one end the mutation may be very mild, whereas at the other end it is very severe similar to homozygous β-thalassemia or thalassemia major. Our approach does not require an antibiotic selection cassette that may interfere with expression of the corrected gene [6, 9, 12]. Cookies policy.

Huang X, Wang Y, Yan W, Smith C, Ye Z, Wang J, et al. PubMed  2010;24(1):27–37.

Darüber hinaus bilden sie die Grundlage der „mole... Time-lapse analysis of HEK293 cells transfected with pExon1Q23-Kaede-A. 2010;12(2):61–76. Seamless gene correction of beta-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Do you all use calcium phosphate methods to tranfect cells? Nat Biotech. Thanks ! Blood. In this study, the anti-human beta hemoglobin antibody could not distinguish the one amino acid difference from abnormal hemoglobin E in the Eβ-iPSC2 cells.

To ... Map and Sequence File: Download Open . b Schematic of targeted region of the HBB locus of the patient with HbE/β-thalassemia. Seems strange that the more DNA you add to the transfection the more dying/dead cells you see. Our transfection efficiency varied among iPSC lines, ranging from 30 to 60%. Total RNA was obtained using TRIzol® reagent (Invitrogen). Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Samples were loaded onto 18% SDS-PAGE and transferred to a PVDF membrane. pSpCas9(BB)-2A-GFP (PX458) Zhang lab plasmid for expressing a chimeric guide RNA (gRNA) plus EGFP and human codon-optimized Cas9. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein.

In addition, our study further demonstrates that these cells can differentiate in vitro to reticulocytes, which can be developed for therapeutic use. 2015;21(2):121–31. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. 2a).

Factor-induced reprogramming and zinc finger nuclease-aided gene targeting cause different genome instability in β-thalassemia induced pluripotent stem cells (iPSCs). Generation and characterization of Eβ-iPSCs from a HbE/β-thalassemic patient’s HDFs were performed as described previously [15]. After genetic correction, all of the five corrected clones were able to differentiate into hematopoietic progenitor cells which expressed CD34 and CD43, and erythroid markers CD71 and CD235a at comparable levels to those of the HDF-iPSCs in both adherent and nonadherent cell population (Fig. The 2A-GFP allows for screening of transfected cells. J Biol Chem. c Quantitative RT-PCR analysis of erythroid-associated transcription factors at day 19 of differentiation (equivalent to day 13 of erythroid liquid culture) of the Eβ-iPSC2 cells and the corrected C46 cells as compared to peripheral blood CD34+ cell-derived erythroblasts at day 13 (PB). HbE/β-thalassemia, a double heterozygosity of hemoglobin E (HbE) and β-thalassemia, is the most common thalassemic syndrome found in adults in Southeast Asia. Nat Biotech. PubMed Google Scholar. Recently, in vitro genetic correction of hematopoietic stem cells (HSCs) has been successfully reported; however, culturing and maintaining HSCs remain difficult [24]. Red box indicates HbE mutation (G → A) at codon 26. 2015;163(3):759–71. Addgene is a nonprofit plasmid repository. Google Scholar. For off-target analysis, five potential off-target sites were amplified by PCR. Nat Protoc.

Our study provides a successful strategy to correct HbE mutation in one step and could be employed as a universal approach in the future correction of the HBB gene in iPSCs derived from other HbE/β0-thalassemia or β+-thalassemia patients.

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