scholarly journals p53-TP53-Induced Glycolysis Regulator Mediated Glycolytic Suppression Attenuates DNA Damage and Genomic Instability in Fanconi Anemia Hematopoietic Stem Cells

Stem Cells ◽  
2019 ◽  
Vol 37 (7) ◽  
pp. 937-947 ◽  
Author(s):  
Xue Li ◽  
Limei Wu ◽  
Morgan Zopp ◽  
Shaina Kopelov ◽  
Wei Du
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Genomic Instability ◽  
Fanconi Anemia ◽  
Hematopoietic Stem

scholarly journals Fanconi Anemia: A Paradigm for Understanding DNA Repair During Replication.

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-32-SCI-32 ◽  
Author(s):  
Agata Smogorzewska
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Dna Repair ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Chromosome Breakage ◽  
Dna Lesions ◽  
Hematopoietic Stem

Fanconi anemia, the most common hereditary bone marrow failure disorder, results from defective repair of DNA interstrand crosslinks (ICLs), which covalently link complementary DNA strands causing replication stalling. Mutations in 22 different genes (FANCA-FANCW) have been shown to result in Fanconi anemia. Their protein products work at different stages of DNA repair leading to considerable heterogeneity in human phenotypes. The majority of the FANC gene mutations are recessively inherited with the exceptions of FANCB and FANCR/RAD51. FANCB is X-linked, and all FANCR/RAD51 mutations arise de novo, affect only one allele, and the mutant protein acts as a dominant negative against the wild type protein. Despite advances in the molecular diagnosis of Fanconi anemia, if Fanconi anemia is suspected, chromosome breakage (DEB or MMC) testing on patient cells is essential. We have seen a number of patients referred to the International Fanconi Anemia Registry (http://lab.rockefeller.edu/smogorzewska/ifar/) who are misdiagnosed with Fanconi anemia based solely on the presence of a FANC gene variant in gene panel or whole exome sequencing. Conversely, blood mosaicism may lead to a negative blood chromosome breakage test. If there is a high suspicion of Fanconi anemia, but blood breakage results are negative, breakage test on patient fibroblasts should be performed. Diagnosis of Fanconi anemia should also be entertained in young adults presenting with squamous cell carcinoma of the aerodigestive tract, since this may be their initial presentation of Fanconi anemia and conventional chemotherapy dose would precipitate bone marrow failure in these patients. In my talk, I will discuss the mechanism of the Fanconi anemia repair pathway during DNA replication. Then, I will concentrate on the mechanism of bone marrow failure and tumorigenesis in Fanconi anemia. I will explore the hypothesis that the endogenously produced aldehydes including some that are still unknown, contribute to disease development. Fanconi anemia-deficient hematopoietic stem cells have an autonomous DNA repair defect. Accumulation of DNA damage leads to apoptosis due to the activation of p53. If cells escape death, mutagenesis may lead to the development of leukemia. The sources of endogenous DNA damage are poorly understood. Cell cycle induction of Fanconi anemia pathway-deficientmouse hematopoietic stem cells results in DNA damage and bone marrow failure, which implies that the DNA lesions encountered during replication are the culprit. There is mounting evidence that the endogenous aldehydes, including acetaldehyde and formaldehyde,may cause those DNA lesions. To identify other metabolites that may induce bone marrow failure in Fanconi anemia, we used a library of CRISPR guides to target Cas9 to metabolic genes to screen for and identify synthetic lethality with Fanconi anemia deficiency. We have identifiedALDH9A1as the most significantly depleted gene in FANCD2-/- cells. The synthetically lethal interaction was validated using single gene editing in human umbilical cord-derived hematopoietic stem progenitor cells. We propose a model in which aldehydes that are metabolized by ALDH9A1 accumulate in the absence of this enzyme and cause DNA damage that requires the Fanconi anemia pathway proteins for repair, survival, and suppression of tumorigenesis. We are testing this model using Fanca-/-Aldh9a1-/-mice. Disclosures No relevant conflicts of interest to declare.

scholarly journals ¬¬Attenuated DNA Damage Responses and Increased Apoptosis Characterize Human Hematopoietic Stem Cells Exposed to Irradiation

2018 ◽  
Vol 64 ◽  
pp. S87
Author(s):  
Michael Milyavsky ◽  
Shahar Biechonski ◽  
Leonid Olender ◽  
Adi Zipin-Roitman ◽  
Muhammad Yassin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Dna Damage Responses

scholarly journals Genistein Protects Hematopoietic Stem Cells against G-CSF–Induced DNA Damage

2014 ◽  
Vol 7 (5) ◽  
pp. 534-544 ◽  
Author(s):  
Liliana R. Souza ◽  
Erica Silva ◽  
Elissa Calloway ◽  
Omer Kucuk ◽  
Michael Rossi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem

Non-Random Lentiviral Vector Insertions in Bone Marrow Progenitors from Fanconi Anemia Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2358-2358
Author(s):  
Ali Nowrouzi ◽  
Africa Gonzales-Murillo ◽  
Anna Paruzynski ◽  
Ariana Jacome ◽  
Paula Rio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Large Scale ◽  
Random Distribution ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
In Cis

Abstract Improved protocols using lentiviral vectors have been established with minimal cytokine exposure and short transduction times proving more suitable for overcoming the disease-specific challenge in correcting functionally defective hematopoietic stem cells (HSCs) of Fanconi Anemia (FA) patients. Bone marrow (BM) cells from FA patients were transduced ex vivo with lentiviral vectors (LVs) expressing FANCA and/or EGFP using optimized conditions to preserve the repopulating properties of the primitive hematopoietic stem cells (manuscript submitted). In a forward preclinical screening of possible LV-induced side effects we analyzed the insertional inventory in colonies generated by FA BM cells previously transduced with the LVs. We have established and optimized DNA and RNA isolation procedures for minimal cell numbers, suitable for large scale screening of colony forming cell (CFC) derived colonies by linear amplification-mediated PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; Roche). This approach is applicable for detecting early indicators of clonal selection, and is based on the analysis of common integration sites (CIS) and non-random distribution of vector insertions in particular genomic loci. From a total of 180 CFC-derived colonies expressing the EGFP LV marker gene, 298 vector insertions could be sequenced and mapped to the human genome. The analysis of vector targeted gene coding regions showed a non-random genomic distribution of LV insertions, with a significant overrepresentation of RefSeq genes that are part of distinct functional categories. Accordingly vector associated genes are predominantly involved in cellular signal cascades regulated by the MAP Kinase family known to be involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Apart from the observed high integration frequency in genes (>80%), partial loss of vector LTR nucleotides was detected in >10% of the integrants (3–25bp). Notably, >20% of the lentiviral insertions were found to be located in CIS of predominantly 2nd order. Further screening assays of LV transduced CFC-derived colonies will allow a deeper investigation in the functional consequences of such CIS targeting in gene therapy protocols of FA. However our results suggest that the LV transduction of FA BM progenitors leads to a relatively high frequency of insertions in CIS which may be indicative of an insertion based (specific) selection mechanism. We herby show that the ex vivo large scale integration site analyses of CFC-derived colonies from patients considered to undergo gene therapeutic treatments constitutes a robust approach, which combined with mouse preclinical biosafety studies will help to improve the safety of clinical gene therapy protocols. The non-random distribution of LV integrations in CIS associated genes and in genes involved in particular cellular pathways may be indicative for the altered biochemical pathways characteristic of FA stem cells, with reported defects in DNA repair and self-renewal.

CML-CP Mouse Model of Genomic Instability.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1210-1210
Author(s):  
Elisabeth Bolton ◽  
Linda Kamp ◽  
Hardik Modi ◽  
Ravi Bhatia ◽  
Steffen Koschmieder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Stem Cell ◽  
Mouse Model ◽  
Board Of Directors ◽  
Genomic Instability ◽  
Oxidative Dna Damage ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Dependent Effect

Abstract Abstract 1210 Background: BCR-ABL1 transforms hematopoietic stem cells to induce chronic myeloid leukemia in chronic phase (CML-CP). Although CML is stem cell-derived, it is a progenitor cell-driven disease. In CML-CP, leukemia stem cells (LSCs) are characterized by elevated BCR-ABL1 expression in comparison to leukemia progenitor cells (LPCs). Increased expression of BCR-ABL1 kinase is also associated with progression from CML-CP to CML-blast phase. Previously we showed that BCR-ABL1 kinase stimulates reactive oxygen species (ROS)-dependent DNA damage resulting in genomic instability in vitro, which was responsible for acquired imatinib-resistance and accumulation of chromosomal aberrations (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). Result: To examine the effects of BCR-ABL1 expression on genomic instability during in vivo leukemogenesis we employed an inducible transgenic mouse model of CML-CP with targeted expression of p210BCR-ABL1 in hematopoietic stem and progenitor cells (Koschmieder et al., Blood, 2005). Mice exhibiting CML-CP-like disease resulting from BCR-ABL1 induction demonstrated splenomegaly, leukocytosis, and Gr1+/CD11b+ myeloid expansion in bone marrow, spleen and peripheral blood, as detected by FACS analysis. BCR-ABL1 mRNA expression was higher in Lin-c-Kit+Sca1+ stem-enriched cells than in Lin-c-Kit+Sca1- progenitor-enriched cells, thus reminiscent of CML-CP (LSCs>LPCs). BCR-ABL1 increased levels of ROS (hydrogen peroxide, hydroxyl radical) and oxidative DNA lesions (8-oxoG) in LSC-enriched Lin-c-Kit+Sca1+ cells. Preliminary data also suggested that quiescent (CFSEmax) Lin-c-Kit+Sca1+ cells from BCR-ABL1-induced mice exhibited greater ROS (superoxide) production than non-induced counter parts. Moreover, higher levels of ROS were detected in BCR-ABL1-positive Lin-c-Kit+Sca1+ stem-enriched population in comparison to BCR-ABL1-positive Lin-c-Kit+Sca1- progenitor population, suggesting a dosage-dependent effect of BCR-ABL1. To confirm that BCR-ABL1 exerts a dosage-dependent effect on ROS-induced oxidative DNA damage, we showed that the levels of ROS, 8-oxoG and DNA double-strand breaks were proportional to BCR-ABL1 kinase expression in murine 32Dc13 and human CD34+ cells. Conclusion: In summary, this mouse model recapitulates the BCR-ABL1 expression profile attributed to stem and progenitor populations in human CML-CP. It also shows that the BCR-ABL1-positive, stem cell-enriched Lin-c-Kit+Sca1+ population displays elevated levels of ROS and oxidative DNA damage in comparison to normal counterparts, which makes it suitable to study the mechanisms of genomic instability in LSCs. Single nucleotide polymorphism (SNP) arrays will shed more light on the genomic instability of this BCR-ABL1-induced transgenic model of CML-CP. Disclosures: Koschmieder: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.

Regulation of FANCL by Glycogen Synthase Kinase-3beta Links the Fanconi anemia pathway to Self Renewal and Survival Signals

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1263-1263
Author(s):  
Kim-Hien T. Dao ◽  
Michael D. Rotelli ◽  
Jane E. Yates ◽  
Brieanna Brown ◽  
Juha Rantala ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Glycogen Synthase ◽  
Tnf Alpha ◽  
Beta Catenin ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Glycogen Synthase Kinase 3Beta ◽  
N Terminus

Abstract Abstract 1263 The molecular basis for how a Fanconi anemia (FA) genetic background contributes to hematopoietic stem cell defects and hypoplastic organ development remains poorly understood. Protein modification by ubiquitination is a mechanism that diversifies the function and regulation of proteins. In light of this, we focus on the dysfunction of FANCL, the E3 ubiquitin ligase of the FA pathway, as a key molecular defect in Fanconi anemia. Here we report our studies investigating mechanisms of post-translational regulation of FANCL. We view these mechanisms as potential targets to augment the function of the FA core complex and correct hematopoietic stem cell defects. We provide evidence that FANCL is exquisitely regulated by ubiquitin-proteosome degradation. Ligase-inactive mutants (FANCL-C307A and -W341G) are less sensitive to this regulation, suggesting a role for auto-ubiquitination in directing lysine-48 polyubiquitination. This constitutive negative regulation of FANCL is partially reversed with an ATP-competitive glycogen synthase kinase-3beta (GSK-3beta) inhibitor. GSK-3beta is a serine/threonine kinase that phosphorylates proteins and marks them for ubiquitin-mediated proteolysis. Mitogenic and survival pathways, including Ras/MAPK and PI3K/Akt, negatively regulate GSK-3beta by serine-9 phosphorylation. We show that the regulation of FANCL by GSK-3beta is likely direct because FANCL and GSK-3beta co-immunoprecipitate in cell lysates and as GST-fusion proteins. To define the biochemical mechanisms of FANCL regulation, we generated N-terminal deletion mutants of FANCL and we show that the regulation of FANCL is dictated by a region at the N-terminus (aa1-78). Mutational analysis of FANCL (lysine to arginine) in this N-terminus region does not affect the overall protein level or ubiquitination of FANCL, suggesting that FANCL may be targeted for degradation by phosphorylation and/or in a complex with other proteins. The potential biological relevance of our findings, that FANCL is regulated by GSK-3beta is revealed in studies overexpressing constitutively active, myristoylated-Akt. This experimental condition increases FANCL protein levels and suggests a role for FANCL as a downstream effector of PI3K/Akt signaling. In turn, FANCL likely regulates non-canonical targets that alter the transcriptome profile favoring self-renewal and survival of hematopoietic stem cells. We recently published our studies identifying beta-catenin as one such downstream target (Blood 2012 Jul 12;120:323). Suppression of FANCL expression severely disrupts Wnt/beta-catenin signaling and expression of downstream Wnt-responsive targets MYC and CCND1. We also identified that GSK3B gene expression is approximately 5-fold higher in Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared to untreated cells or to wildtype cells with or without TNF-alpha. Our current studies show that inhibition of GSK-3beta preserves the number of murine Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared with no GSK-3beta inhibition. Taken together, we have accumulated evidence suggesting that GSK-3beta is a promising molecular target to improve the self-renewal and survival of FA hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genomic Instability Originates From Leukemia Stem Cells in a Mouse Model of CML-CP

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 445-445 ◽  
Author(s):  
Elisabeth Bolton ◽  
Mirle Schemionek ◽  
Hans-Ulrich Klein ◽  
Linda Kerstiens ◽  
Steffen Koschmieder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Genomic Instability ◽  
Oxidative Dna Damage ◽  
Transgenic Mouse Model ◽  
Hematopoietic Stem ◽  
Genetic Aberrations ◽  
Stem And Progenitor Cells

Abstract Abstract 445 For decades, chronic myeloid leukemia (CML) has served not only as a paradigm for understanding the evolution and multi-step process of carcinogenesis but also for studying cancer stem and progenitor cells responsible for the initiation and/or maintenance of the disease. CML is initiated by BCR-ABL1 tyrosine kinase transformation of hematopoietic stem cells into leukemia stem cells (LSCs) to induce CML-chronic phase (CML-CP). The deregulated growth of LSC-derived leukemia progenitor cells (LPCs) leads to manifestation of the disease. It is unclear if LSCs and/or LPCs are able to acquire additional genetic changes that confer resistance to tyrosine kinase inhibitors (TKIs) and induce more aggressive CML blast phase (CML-BP). In addition, the mechanisms and consequences of genomic instability may differ substantially among these cells. For example, the effects of genetic aberrations acquired in quiescent LSCs may be dormant, but if the aberrations induce proliferation or appear in LSCs that are already cycling, they may generate TKI-resistant and/or more malignant clones. Alternatively, genomic instability in LPCs must be accompanied by the acquisition of LSC-like properties to prevent mutations from disappearing before they undergo terminal maturation. Previously, we reported that BCR-ABL1–transformed cell lines accumulate reactive oxygen species (ROS)-induced oxidative DNA damage [8-oxoguanine (8oxoG), double strand breaks (DSBs)] resulting in genomic instability in vitro, which was responsible for acquired imatinib-resistance and accumulation of chromosomal aberrations (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). To determine which populations of CML-CP cells, LSCs and/or LPCs, accumulate genomic instability we employed the SCLtTA/BCR-ABL1 tetracycline-inducible (tet-off) transgenic mouse model of CML-CP with targeted expression of p210BCR-ABL1 in hematopoietic stem and progenitor cells (Koschmieder et al., Blood, 2005). Mice exhibiting CML-CP-like disease resulting from BCR-ABL1 induction demonstrated splenomegaly and Gr1+/CD11b+ myeloid expansion in bone marrow, spleen and peripheral blood. BCR-ABL1 mRNA expression was higher in the Lin−c-Kit+Sca1+ murine leukemia stem cell–enriched population (muLSCs) than in the Lin−c-Kit+Sca1− murine leukemia progenitor cell–enriched population (muLPCs), thus reminiscent of human CML-CP (Lin−CD34+CD38− LSCs > Lin−CD34+CD38+ LPCs). BCR-ABL1 induction increased levels of ROS (hydrogen peroxide, hydroxyl radical) and oxidative DNA damage (8-oxoG, DSBs) in muLSCs, but not in muLPCs. In addition, CFSEmax/eFluor670max quiescent muLSCs displayed more ROS (superoxide, hydrogen peroxide) and oxidative DNA damage (8oxoG, DSBs) than non-induced counterparts. Currently, we are examining genomic instability in the most primitive long-term muLSCs (Lin−c-Kit+Sca1+CD34−Flt3−). Lastly, single nucleotide polymorphism (SNP) arrays detected a variety of genetic aberrations (addition, deletions) in BCR-ABL1–induced Lin− BM cells. Individual mice displayed a great degree of diversity in the intensity of genetic instability accumulating between 31 to 826 aberrations, which recapitulate heterogeneity of sporadic aberrations detected in CML-CP patients. These aberrations include deletions in Trp53 and Ikzf1, and additions in Zfp423 and Idh1 genes, which have been linked to progression from CML-CP to CML-BP. In summary, by using the SCLtTA/BCR-ABL1 inducible transgenic mouse model of CML-CP we showed that muLSCs, but not muLPCs, displayed elevated levels of ROS-induced oxidative DNA damage likely resulting in the accumulation of extensive genetic aberrations. This observation supports the hypothesis that genomic instability in CML-CP originates in LSCs. Current analysis of microarrays may shed some light on the mechanisms leading to enhanced ROS production and accumulation of oxidative DNA damage in muLSCs. Disclosures: Koschmieder: Novartis, Bristol-Myers Squibb: Consultancy.

scholarly journals A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1108-1108
Author(s):  
Masoud Nasri ◽  
Perihan Mir ◽  
Benjamin Dannenmann ◽  
Diana Amend ◽  
Yun Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Gene Editing ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematopoietic Stem ◽  
Editing Efficiency ◽  
Human Ipscs

Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document