Enhancement of red blood cell transfusion compatibility using CRISPR‐mediated erythroblast gene editing
2018; Springer Nature; Volume: 10; Issue: 6 Linguagem: Inglês
10.15252/emmm.201708454
ISSN1757-4684
AutoresJoseph Hawksworth, Timothy J. Satchwell, Marjolein Meinders, Deborah E. Daniels, Fiona Regan, Nicole Thornton, Marieangela C. Wilson, Johannes G. G. Dobbe, Geert J. Streekstra, Kongtana Trakarnsanga, Kate J. Heesom, David J. Anstee, Jan Frayne, Ashley M. Toye,
Tópico(s)Parvovirus B19 Infection Studies
ResumoReport26 April 2018Open Access Transparent process Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing Joseph Hawksworth School of Biochemistry, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Timothy J Satchwell School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Marjolein Meinders School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author Deborah E Daniels School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Search for more papers by this author Fiona Regan Imperial College Healthcare NHS Trust, London, UK NHS Blood & Transplant, London, UK Search for more papers by this author Nicole M Thornton International Blood Group Reference Laboratory, National Health Service (NHS) Blood and Transplant, Bristol, UK Search for more papers by this author Marieangela C Wilson School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author Johannes GG Dobbe Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Geert J Streekstra Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Kongtana Trakarnsanga Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Search for more papers by this author Kate J Heesom School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author David J Anstee School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Jan Frayne School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Search for more papers by this author Ashley M Toye Corresponding Author [email protected] orcid.org/0000-0003-4395-9396 School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Joseph Hawksworth School of Biochemistry, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Timothy J Satchwell School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Marjolein Meinders School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author Deborah E Daniels School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Search for more papers by this author Fiona Regan Imperial College Healthcare NHS Trust, London, UK NHS Blood & Transplant, London, UK Search for more papers by this author Nicole M Thornton International Blood Group Reference Laboratory, National Health Service (NHS) Blood and Transplant, Bristol, UK Search for more papers by this author Marieangela C Wilson School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author Johannes GG Dobbe Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Geert J Streekstra Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Kongtana Trakarnsanga Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Search for more papers by this author Kate J Heesom School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author David J Anstee School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Jan Frayne School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Search for more papers by this author Ashley M Toye Corresponding Author [email protected] orcid.org/0000-0003-4395-9396 School of Biochemistry, University of Bristol, Bristol, UK NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK Search for more papers by this author Author Information Joseph Hawksworth1,3,‡, Timothy J Satchwell1,2,3,‡, Marjolein Meinders1, Deborah E Daniels1,2, Fiona Regan4,5, Nicole M Thornton6, Marieangela C Wilson1, Johannes GG Dobbe7, Geert J Streekstra7, Kongtana Trakarnsanga8, Kate J Heesom1, David J Anstee1,2,3, Jan Frayne1,2 and Ashley M Toye *,1,2,3 1School of Biochemistry, University of Bristol, Bristol, UK 2NIHR Blood and Transplant Research Unit, University of Bristol, Bristol, UK 3Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Bristol, UK 4Imperial College Healthcare NHS Trust, London, UK 5NHS Blood & Transplant, London, UK 6International Blood Group Reference Laboratory, National Health Service (NHS) Blood and Transplant, Bristol, UK 7Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 8Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand ‡These authors contributed equally to this work *Corresponding author. Tel: +44 0117 3312111; E-mail: [email protected] EMBO Mol Med (2018)10:e8454https://doi.org/10.15252/emmm.201708454 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle-cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Fynull), GPB (S−s−U−). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements. Synopsis CRISPR–Cas9-mediated genome editing of immortalised erythroblasts is a viable approach to the generation of functional in vitro derived reticulocytes with customised depletion of antigenic blood group proteins that could ultimately facilitate transfusion of patients with unmet clinical needs. Sustainable immortalised erythroblast lines depleted for individual blood groups can be generated using CRISPR–Cas9 gene editing for serological diagnostics. Multiple blood group genes can be knocked out to generate multi-compatible RBCs with future potential to service the unmet clinical transfusion needs of patients with complicated matching requirements. Five blood group null phenotypes are able to coexist in differentiating erythroblasts for the generation of functional reticulocytes. Introduction The collection of more than 1.5 million units of blood is required each year to meet the transfusion needs of England alone (MHRA, 2016). For the majority of patients, this need is serviced by the blood donation system which provides hospitals with screened donor blood that is matched for the major blood group antigens: A, B, O and RhD. Donors who are blood group O RhD negative are often described as "universal donors"; however, this popular simplification misrepresents the complexity of RBC surface antigens that influence donor–recipient compatibility with 36 blood group systems and more than 350 different antigens recognised by the International Society of Blood Transfusion (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/). Mismatch of any blood group antigen has the potential to cause alloimmunisation (generation of antibodies to non-self RBC antigens). Across all transfusion recipients, this occurs in approximately 2–5% of cases; however, in chronically transfused sickle-cell disease (SCD) patients, RBC alloimmunisation occurs in approximately 30% of cases (Campbell-Lee & Kittles, 2014). Common alloantibodies generated in chronically transfused patients with SCD include those to the C and E antigens of the Rh blood group system (RH), K in the Kell system (KEL), Fya and Fy3 in the Duffy system (FY), Jkb in the Kidd system (JK), and U and S in the MNS blood group system (Rosse et al, 1990; Aygun et al, 2002; Castro et al, 2002). Such incompatibilities often result from the differing prevalence of antigens between SCD recipients of African descent compared to a predominately Caucasian donor base. Whilst alloimmunisation of chronically transfused patients increases the difficulty in obtaining suitably matched donations, individuals exist with non-pathological but particularly rare naturally occurring blood group phenotypes that also present major challenges for blood transfusion services across the world to match. Examples include individuals with the rare Bombay phenotype [one in 250,000 of the population worldwide (Mallick et al, 2015) and one in 1,000,000 in Europe (Oriol et al, 2000)] who lack the H antigen—the antigenic determinant of blood group O and the precursor of the A and B antigens—and those who are Rhnull [one in every 6,000,000 (Avent & Reid, 2000)] who lack all antigens encoded by both the RHD and RHCE genes. In these, and other rare cases, transfusion with only ABO RhD matched blood will result in adverse effects and therefore donations are required from an individual with the same rare phenotype. Current efforts to cater for individuals with rare blood types rely on international coordination of rare donor databases and the cryopreservation of donor units including those stored for autologous transfusion (Anstee et al, 1999; Nance, 2009; Nance et al, 2016).The ability to generate RBCs with bespoke phenotypes for individuals with rare blood types together with a "more universal" source of RBCs designed to minimise alloimmunisation in SCD patients and to meet the transfusion needs of existing patients for whom alloimmunisation has reduced the suitable donor pool would have obvious clinical benefits. The desire to improve RBC compatibility for transfusion is not a new concept. Whilst the successful conversion of blood group types A and B to O has been demonstrated using glycosidases (Zhu et al, 1996; Kruskall et al, 2000; Liu et al, 2007), attempts to more broadly enhance compatibility through antigen masking achieved only limited success (Jeong & Byun, 1996; Armstrong et al, 1997; Scott et al, 1997). More recently, developments in the in vitro culture of erythroid cells have facilitated the laboratory proof-of-principle production of RBCs with severe depletion in the expression of other blood groups using lentiviral expression of shRNAs in haematopoietic stem cells (Bagnis et al, 2009; Cambot et al, 2013). In some cases, these modified cells present the properties of their equivalent null phenotypes as assessed by routine diagnostic haemagglutination tests (Bagnis et al, 2009; Cambot et al, 2013) although remaining residual antigens resulting from incomplete knockdown may still result in alloantibody formation in vivo. An additional obstacle to the use of adult haematopoietic stem cells for the culture of modified RBCs is presented by the finite proliferative capacity of such cells, which currently limits the yield of cultured RBCs to sub-transfusable quantities and the requirement for repeated shRNA transduction between cultures. Alternative approaches which have focused on the derivation of RBCs from more sustainable induced pluripotent stem cells created from existing donors with rare or more universally acceptable RBC phenotypes such as Bombay have been confounded by poor levels of erythroid cell expansion and aberrant or incomplete differentiation (Seifinejad et al, 2010). The generation of immortalised erythroid cell lines provides potential for an unlimited supply of red cells. Gene editing technologies can be utilised in these lines to make modifications which improve transfusion compatibility without the need for repeated editing of haematopoietic stem cells (Kim et al, 2015). In this study, we use CRISPR-mediated genome editing of a recently developed immortalised human erythroblast cell line BEL-A (Trakarnsanga et al, 2017) to generate stable erythroblast cell lines that can be differentiated to generate functional reticulocytes completely deficient in a variety of individual transfusion-relevant blood groups. By simultaneously expressing multiple guide RNAs (gRNAs) in these cells, we demonstrate the ability to delete multiple blood group genes in erythroblasts and present proof-of-principle generation of red blood cells completely deficient in blood groups encoded by five different genes that encode antigens responsible for the most common transfusion incompatibilities. Results Target antigen selection In order to direct the design of a bespoke or customised RBC phenotype that would be able to service existing unmet clinical transfusion needs within the blood transfusion service in England (NHSBT), a survey was conducted to collate instances where rare or problematic transfusion requirements were identified and in which the requirement for matched blood could not be fulfilled or resulted in no remaining store for subsequent patients. This survey, encompassing a combined 15 month period (November 2014–January 2015; April 2015–April 2016), identified 56 patients with rare blood types with alloantibodies against RBC antigens (or with the likelihood of acquisition without specific matching, e.g. in the case of untransfused neonates). Table 1 summarises the data collected in this study; 22 patients had alloantibodies to antigens located on glycophorin B (GPB), U, S or s; 19 patients presented with alloantibodies to at least one antigen within the Rh blood group system; 10 patients possessed alloantibodies to the Duffy blood group [with a further two previously untransfused patients also Fy(a−b−)]; Kell antigen alloantibodies were identified in 10 patients. Eight patients presented with the Bombay or para-Bombay phenotype (a rare phenotype in which a mutation in the FUT1 gene results in the loss of the H antigen and transfusion incompatibility with all ABO blood), and alloantibodies to Lu and Kidd antigens were identified in three patients for each, respectively. In total, 19 patients (the majority of them presenting with SCD) possessed alloantibodies to antigens within more than one blood group system. Table 1. Identification of clinical need within NHSBT for rare erythrocyte phenotypes Blood group system Patients with alloantibodies Alloantibodies directed against antigens Genetic basis of antigens MNS 22 U, S, s GYPB Rh 19 D, C, E, c, e, CW, Hr0, hrB, HrB, MAR RHD, RHCE Duffy 10 (+2) Fya, Fyb, Fy3 ACKR1 Kell 10 K, k, Kpa KEL H 8 H FUT1 Lutheran 3 Lua, Lub BCAM Kidd 3 Jkb SLC14A1 Results of a survey collating instances in which rare or problematic transfusion requirements were identified and in which the requirement for matched blood could not be fulfilled or resulted in no remaining store for subsequent patients. November 2014–January 2015; April 2015–April 2016. Fifty-six patients in total, (+2) indicates untransfused individuals of Fy(a−b−) phenotype yet to develop alloantibodies. Two patients with McLeod syndrome (XK deficiency) are not listed. This study highlights the major blood groups for which alloimmunisation and transfusion incompatibility is most relevant within the population serviced by NHSBT. Discounting the two McLeod patients [in which absence of XK results in the undesirable trait of acanthocytic erythrocytes (Wimer et al, 1977)], the transfusion requirements of 54 of the 56 patients identified could be serviced by a hypothetical RBC phenotype lacking 7 blood group proteins and 48 of these patients by the removal of just 5. On this basis, we decided to pursue the generation of RBCs completely deficient in surface expression of GPB, Rh, Kell, Duffy and of the Bombay phenotype (able to be received by recipients of blood group A, B, AB, O or Bombay) using CRISPR–Cas9 editing for gene knockout in a recently published immortalised erythroblast cell line: BEL-A (Trakarnsanga et al, 2017). Generation of individual blood group knockout cell lines for transfusion therapy and as tools for diagnostics In addition to acting as a much-needed source of rare blood for transfusion requirements, blood donations by patients with unusual phenotypes play a crucial role in serological testing by blood group reference laboratories. These so-called reagent red cells with established complete deficiency of a given blood group or antigen, for example Rhnull or K0, provide essential controls that facilitate the identification of unknown alloantibodies in patient sera. Such cells, currently curated and stored frozen by selected International Blood Group Reference Laboratories, represent a finite resource, reliant upon the identification of and donations from individuals with often extremely rare naturally occurring mutations and phenotypes. The ability to generate a sustainable library of enucleation competent immortalised erythroblast cell lines completely deficient in expression of a range of individual blood groups of interest would have obvious benefits for serological testing laboratories. As a first step towards the combinatorial removal of multiple blood groups and for the generation of cell lines with potential therapeutic and diagnostic applications, we sought to generate BEL-A cell lines with individual blood group knockouts useful for these purposes. BEL-A cells were first single cell sorted by fluorescence-activated cell sorting (FACS) and expanded to derive a founder population, expanding cells were lentivirally transduced with a construct containing Cas9 and gRNA targeting the gene of interest as described in Materials and Methods. Following transduction, cells were maintained in culture for at least 1 week to enable turnover of existing protein, labelled with a specific antibody to the extracellular epitope of the blood group protein targeted and then single cell sorted by FACS based on complete negativity for protein of interest. In the case of GPB, in which the protein was not expressed in undifferentiated cells, blind sorting was conducted to derive single clones. These clones were subsequently differentiated and analysed by flow cytometry to confirm the absence of GPB surface expression in reticulocytes. Since the founder BEL-A cell line was derived from a donor of Rh type D+C−c+E+e+ (for full blood group genotype see Table 2), both RhD and RhCE required removal. To simplify this, a single guide was designed to target RHAG, the gene encoding the Rh-associated glycoprotein, essential for stable surface expression of Rh and in which mutations are known to result in Rhnull (regulator type) erythrocytes (Cherif-Zahar et al, 1996; Huang, 1998). Individual blood group knockout BEL-A clones were expanded and a proportion differentiated for verification of null phenotypes in enucleated reticulocytes. Figure 1 shows the complete absence of expression of proteins targeted by CRISPR gRNAs in knockout reticulocytes compared to reticulocytes derived from unedited cells, as assessed by flow cytometry. In each case, reticulocytes were also labelled with a panel of antibodies to additional major erythrocyte membrane proteins: band 3, GPA, GPC and CD47 (Fig EV1). The anticipated reduction in CD47, previously reported for Rhnull erythrocytes (Mouro-Chanteloup et al, 2003), was recapitulated whilst expression of band 3, GPA and GPC was unaltered compared to untransduced control cells. Table 2. BEL-A genotype Blood group system BEL-A predicted phenotype Rh (RH) D+C−c+E+e+ Duffy (FY) Fy(a+b−) Kidd (JK) Jk(a−b+) Kell (KEL) K−k+ MNS (MNS) M−N+S−s+ Lutheran (LU) Lu(a−b+) Diego (DI) Di(a−b+) Colton (CO) Co(a+b−) Dombrock (DO) Do(a+b−) Landsteiner–Wiener (LW) LW(a+b−) Scianna (SC) Sc1+Sc2− PCR was used to determine the BEL-A genotype for various blood group antigens. Genotyping was performed by the International Blood Group Reference Laboratory.11 Correction added on 9 May 2018 after first online publication: the blood group nomenclature in Table 2 has been corrected. Figure 1. Flow cytometric confirmation of individual blood group knockouts in BEL-A-derived reticulocytesBEL-A blood group knockout lines were created using lentiviral CRISPR–Cas9. Knockout lines were single cell sorted into clonal sub-lines which were differentiated for 14 days. Enucleated reticulocytes were identified based on negativity for Hoechst 33342. Expression levels of targeted blood group antigens in knockout lines overlay with IgG controls indicating complete protein knockouts. RHAG knockout was screened with both LA1818 (anti-RhAG) and BRIC 69 (anti-Rh) in order to confirm RHAG knockout and Rhnull phenotype. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Flow cytometric analysis of major erythrocyte membrane proteins in individual blood group knockout BEL-A reticulocytesNo unexpected alterations in expression of band 3, GPA, GPC, RhAG or Rh proteins compared to untransduced BEL-A controls were observed. As expected, CD47 expression was reduced in the RhAG knockout line due to disruption of the Rh subcomplex. Download figure Download PowerPoint Combinatorial CRISPR editing for generation of RBC with multiple blood group proteins ablated Having determined guide sequences that enable the successful knockout of each of the blood groups of interest, clonal GPB null BEL-A cells were used as a starting point to generate a cell line transduced with multiple guides resulting in a multiple knockout. GPB null cells were transduced with three lentiCRISPRv2 lentiviruses simultaneously, each containing a guide targeting either KEL (Kell gene), ACKR1 (Duffy gene) or FUT1 (gene encoding fucosyltransferase 1, the enzyme required for the generation of the H antigen). Cells were immunolabelled with antibodies specific for each targeted protein, a triple null population was single cell sorted, and cells were expanded and differentiated for verification of the null phenotype in reticulocytes as described for single knockouts. Cells deficient in GPB, H, Kell and Duffy were subsequently transduced with lentiCRISPRv2 containing a guide targeting RHAG to produce a 5× knockout (KO) BEL-A line. Biallelic mutations in each of the genes targeted were confirmed and are listed in Table EV1. Reticulocytes were generated by in vitro culture, 5× KO and control cells were induced to undergo differentiation and after 14 days reticulocytes were isolated by leukofiltration. Figure 2A shows flow cytometry histograms illustrating the absence of GPB (U and s antigen on a S− background), Kell, Duffy, H antigen, RhAG and Rh (RhCE/D). Cytospins were prepared, and 5× KO and control cells were observed to be morphologically indistinguishable (Fig 2B). Null phenotypes were confirmed by indirect antiglobulin tests (IATs) using human sera containing alloantibodies to antigens in each blood group as indicated (Fig 2C). An extended figure depicting additional RBC controls can be viewed in Fig EV2. Labelling with a panel of antibodies to other proteins identified the expected reduction in CD47 (Mouro-Chanteloup et al, 2003) but no alteration in levels of band 3, GPA or GPC (Fig EV3) confirming the absence of gross membrane disruption. In order to determine whether removal of the five blood group proteins altered the physical characteristics of reticulocytes, an Automated Rheoscope and Cell Analyser (ARCA) was used to assess deformability. The deformability index of 5× KO reticulocytes was found to be only mildly reduced compared to untransduced control reticulocytes (Fig 2D). Figure 2. Characterisation of 5× KO BEL-A reticulocyte phenotypeThe 5× KO BEL-A cell line was created using lentiviral CRISPR–Cas9 targeted to five blood group genes: KEL, RHAG, ACKR1, FUT1 and GYPB. Knockout cells were sorted into clonal sub-lines which were differentiated for 14 days to generate reticulocytes for analysis. Hoechst-negative untransduced BEL-A-derived reticulocytes are positively labelled by antibodies to indicated blood groups/antigens. 5× KO reticulocytes labelled with antibodies to targeted blood group proteins/antigens are completely deficient in expression. Representative cytospin images illustrate similar morphology of leukofiltered reticulocytes derived from untransduced and 5× KO BEL-A cell lines. Indirect antiglobulin test using column agglutination of BEL-A reticulocytes. Absence of GPB, H antigen, Duffy, Kell and Rh in 5× KO reticulocytes is supported by IAT tests with anti-U, anti-H, anti-Fy3, anti-Ku and anti-Rh29 antibodies, respectively. In contrast to the untransduced control cells, 5× KO cells did not agglutinate upon exposure to any of the tested antibodies and cell pellets were observed at the bottom of the microtubules in all tests.22 Correction added on 9 May 2018 after first online publication: the label of the serum control in panel C has been corrected. Deformability index of untransduced control and 5× KO BEL-A cell line-derived reticulocytes determined using an Automated Rheoscope Cell Analyser. Untransduced BEL-A control n = 11, 5× KO n = 5. Error bars indicate standard deviation. Scatter plot depicting relative protein abundance of membrane and cytoskeletal proteins in reticulocytes derived from 5× KO compared to untransduced BEL-A cells as identified by TMT labelling and mass spectrometry. Data were categorised to identify membrane and cytoskeletal proteins using Proteome Discoverer 2.1. Log2 fold ratios are based on the mean of two technical replicates. Data were filtered using a FDR of 1% with exclusion of proteins for which only a single peptide was detected. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Extended serological analysis of 5× KO BEL-A reticulocytesGel card indirect antiglobulin tests support the absence of GPB, H antigen, Kell, Rh and Duffy in 5× KO cells using anti-U, anti-H, anti-Ku, anti-Rh29 and anti-Fy3, respectively. Cellular controls included unedited BEL-A reticulocytes, positive control RBCs and negative control RBCs. AB serum controls were performed with both anti-mouse and anti-human secondary antibodies. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Flow cytometric analysis of major erythrocyte membrane proteins in 5× knockout BEL-A reticulocytesNo alteration in levels of band 3, GPA or GPC confirms the absence of gross membrane disruption. As expected, CD47 expression was reduced due to disruption of the Rh subcomplex. Download figure Download PowerPoint For complete protein level assessment of 5× KO cells, quantitative proteomic analysis was performed using tandem mass tag (TMT) labelled reticulocyte samples. Anticipated reductions in the Rh complex proteins CD47 and ICAM-4 (39 and 93% reductions, respectively) and the Kell interacting protein XK (37% reduction) were confirmed. However, only 2% of membrane and cytoskeletal proteins showed at least a twofold reduction in abundance, demonstrating mi
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