Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
Open accessAbstract
Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols. A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.
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It reports that ribonucleoprotein delivery gives a brief pulse of editing machinery, producing efficient editing with fewer off-target effects.
“RNP delivery results in an initially high level of the genome editing machinery followed by rapid decay, leading to a “fast on, fast off” strategy that results in highly efficient editing while minimizing OTEs13–19.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [13]
The Discussion (page unavailable) describes RNP electroporation as a transient “hit-and-run” approach that enables efficient editing and reduces concerns about off-target cleavage. The paper does not establish the claimed initially high level followed by rapid decay of editing machinery, or demonstrate that this time course causes fewer off-target effects. (LLM, conf=0.85)
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“With the optimized sgRNA delivery method in hand, we next considered whether Cas9 protein pre-complexed with sgRNA to generate a ribonucleoprotein particle (RNP) (Kim et al., 2014; Lin et al., 2014; Schumann et al., 2015) could also be used to edit genes in murine HSPCs.”
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“Several delivery methods have been used to perform CRISPR/Cas9mediated gene editing of HSPCs, including lentiviral transduction (Heckl et al., 2014), plasmid DNA transfection (Mandal et al., 2014), or chemically modified RNA (Hendel et al., 2015), achieving up to 48% gene disruption in human HSPCs.”
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“DNA templates for sgRNAs were made using the protocol described by Li et al (Li et al., 2013) using primers listed in Table S1.”
MethodGenomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting
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“After electroporation of Cas9-expressing c-kit+ cells with GFP-sg1, we sorted CD150+CD48−lineage−Sca-1+ cells (c-kit expression was attenuated within 1 hour of preculture before electroporation, Figure S1D) into semi-solid media and monitored colonies arising from these single HSCs for GFP ablation.”
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“, 2014), plasmid DNA transfection (Mandal et al., 2014), or chemically modified RNA (Hendel et al.”
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“Thus, CRISPR/Cas9 provides a versatile, modular, and cost-effective means to edit the genomes of multiple model systems (Hsu et al., 2014; Sternberg and Doudna, 2015).”
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“Different amounts of GFP-sg1 were mixed with Cas9 protein and the RNP complex was electroporated into HSPCs isolated from a mouse strain that ubiquitously expresses GFP (Schaefer et al., 2001).”
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“After electroporation of Cas9-expressing c-kit+ cells with GFP-sg1, we sorted CD150+CD48−lineage−Sca-1+ cells (c-kit expression was attenuated within 1 hour of preculture before electroporation, Figure S1D) into semi-solid media and monitored colonies arising from these single HSCs for GFP ablation.”
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“It has been shown that monoalleleic loss, but not biallelic loss, of Eed or Suz12 confers proliferative advantages to HSPCs (Lee et al., 2015; Xie et al., 2014).”
BackgroundInfluentialSelf-citationComparison of human cord blood engraftment between immunocompromised mouse strains
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“As expected for this time point (Goyama et al., 2015; McDermott et al., 2010), engrafted human HSPCs gave rise mainly to B- and myeloid cells in the bone marrow in both Cas9 only and Cas9/hCD45-sg1 RNP engrafted mice, with no differences between Gundry et al.”
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Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice
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“Numerous genetically engineered mouse models have contributed to our understanding of the pathways that control HSC maintenance and regeneration in physiological settings (Rossi et al., 2012).”
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“This approach has been used to screen for both positive and negative regulators of HSC function from up to 100 candidate genes (Deneault et al., 2009; Hope et al., 2010).”
MethodA Functional Screen to Identify Novel Effectors of Hematopoietic Stem Cell Activity
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Xenograft models for normal and malignant stem cells
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“As expected for this time point (Goyama et al., 2015; McDermott et al., 2010), engrafted human HSPCs gave rise mainly to B- and myeloid cells in the bone marrow in both Cas9 only and Cas9/hCD45-sg1 RNP engrafted mice, with no differences between Gundry et al.”
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“It has been shown that monoalleleic loss, but not biallelic loss, of Eed or Suz12 confers proliferative advantages to HSPCs (Lee et al., 2015; Xie et al., 2014).”
BackgroundInfluentialHematopoietic stem cell engineering at a crossroads
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“On the other hand, somatic engineering of HSPC genomes has been achieved, in large, using retroviral vectors to either overexpress or to knock-down the expression of genes of interest (Riviere et al., 2012).”
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DNA Damage: From Threat to Treatment
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“DNA templates for 925 sgRNAs contain a T7 promoter, the protospacer sequence, and the sgRNA scaffold 926 sequence (93).”
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Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein
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“4% at 17 weeks after xenotransplantation (Figure 3C), similar to what has been reported previously.(14,23,28) Notably, the 13-nt HPFH mutation occurred at similar frequencies in CD34(1) HSPCs before and after transplantation (Figure 3C; supplemental Figure 3).”
ResultCAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression
Fousek, K., Watanabe, J., Joseph, S. K., George, A., An, X., Byrd, T. T., Morris, J. S., Luong, A., Martinez-Paniagua, M. A., Sanber, K. S., et al. (2020) · Leukemia · doi:10.1038/s41375-020-0792-2 · Cited by 183 · see citing works
Self-citationCarlson-Stevermer, J., Abdeen, A. A., Kohlenberg, L., Goedland, M., Molugu, K., Lou, M., & Saha, K. (2017) · Nature Communications · doi:10.1038/s41467-017-01875-9 · Cited by 170 · see citing works
m6A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment
Cheng, Y., Luo, H., Izzo, F., Pickering, B. F., Nguyen, D. T. T., Myers, R. M., Schurer, A., Gourkanti, S., Brüning, J. C., Vu, L., et al. (2019) · Cell Reports · doi:10.1016/j.celrep.2019.07.032 · Cited by 171 · see citing works
Jayavaradhan, R., Pillis, D. M., Goodman, M., Zhang, F., Zhang, Y., Andreassen, P. R., & Malik, P. (2019) · Nature Communications · doi:10.1038/s41467-019-10735-7 · Cited by 164 · see citing works
CD7 CAR T Cells for the Therapy of Acute Myeloid Leukemia
Gomes‐Silva, D., Atilla, E., Atilla, P. A., Mo, F., Tashiro, H., Srinivasan, M., Lulla, P., Rouce, R. H., Cabral, J. M. S., Ramos, C. A., et al. (2018) · Molecular Therapy · doi:10.1016/j.ymthe.2018.10.001 · Cited by 151 · see citing works
“T cells were activated by plate-bound OKT3 and anti-CD28 antibodies, and the CD7 gene was removed using CRISPR/Cas9 before transduction as previously described.(22,45) In brief, T cells were electroporated with 0.”
MethodTregopathies: Monogenic diseases resulting in regulatory T-cell deficiency
Cepika, A., Sato, Y., Liu, J. M.-H., Uyeda, M. J., Bacchetta, R., & Roncarolo, M. G. (2018) · Journal of Allergy and Clinical Immunology · doi:10.1016/j.jaci.2018.10.026 · Cited by 139 · see citing works
“Follow-up studies(207) used CRISPR-Cas9 editing in HSCs.(208) A second gene-editing strategy used CRISPR-Cas9 ribonucleoproteins with a chemically modified guide RNA(209) and AAV6-mediated homology-directed repair (HDR), with proof of concept for b-hemoglobinopathies.”
Lattanzi, A., Meneghini, V., Pavani, G., Amor, F., Ramadier, S., Félix, T., Antoniani, C., Masson, C., Alibeu, O., Lee, C. M., et al. (2018) · Molecular Therapy · doi:10.1016/j.ymthe.2018.10.008 · Cited by 137 · see citing works
Precancer Atlas to Drive Precision Prevention Trials
Spira, A. E., Yurgelun, M. B., Alexandrov, L. B., Rao, A., Bejar, R., Polyák, K., Giannakis, M., Shilatifard, A., Finn, O. J., Dhodapkar, M., et al. (2017) · Cancer Research · doi:10.1158/0008-5472.can-16-2346 · Cited by 130 · see citing works
Barriga, F. M., Tsanov, K. M., Ho, Y.-J., Sohail, N., Zhang, A., Baslan, T., Wuest, A. N., Del Priore, I., Meškauskaitė, B., Livshits, G., et al. (2022) · Nature Cancer · doi:10.1038/s43018-022-00443-5 · Cited by 125 · see citing works
Gene Therapy for β-Hemoglobinopathies
Cavazzana, M., Antoniani, C., & Miccio, A. (2017) · Molecular Therapy · doi:10.1016/j.ymthe.2017.03.024 · Cited by 124 · see citing works
In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway
Wienert, B., Shin, J. J., Zelin, E., Pestal, K., & Corn, J. E. (2018) · PLoS Biology · doi:10.1371/journal.pbio.2005840 · Cited by 117 · see citing works
CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects
Zhang, Z., Zhang, Y., Gao, F., Han, S., Cheah, K. S. E., Tse, H., & Lian, Q. (2017) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2017.09.009 · Cited by 116 · see citing works
“Gundry et al.(108) remarkably enhanced the genome-editing efficiency of CRISP/Cas9 in human HSCs using purified gRNA and Cas9 nuclease.”
CRISPR/Cas9 gene editing for curing sickle cell disease
Park, S. H., & Bao, G. (2021) · Transfusion and Apheresis Science · doi:10.1016/j.transci.2021.103060 · Cited by 110 · see citing works
“Isolated CD34+ cells were cultured in pre-stimulation media with cytokines for several days before gene-editing, as post-isolation culture with cytokine exposure has been shown to increase the gene-editing efficiency [47].”
MethodPriming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting
Charlesworth, C. T., Camarena, J., Cromer, M. K., Vaidyanathan, S., Bak, R. O., Carte, J. M., Potter, J., Dever, D. P., & Porteus, M. H. (2018) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2018.04.017 · Cited by 100 · see citing works
“Prior work demonstrated that the Cas9/sgRNA system delivered as a RNP complex by electroporation is the most effective method for creating DSBs and stimulating HR in HSPCs.(5,6,19,20) We first sought to optimize the delivery of the Cas9/sgRNA RNP complex to maximize the number of on-target DSBsmade in HSPCs while mini…”
MethodInfluentialPharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells
Kath, J., Du, W., Pruene, A., Braun, T., Thommandru, B., Turk, R., Sturgeon, M. L., Kurgan, G., Amini, L., Stein, M., et al. (2022) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2022.03.018 · Cited by 95 · see citing works
“These differences may result from changes resulting in the incremental developments in our protocols, such as the reduction of the co-transfected RNP amount, as well as the plate-bound stimulation.(25) Notably, the latter potentially reduces”
BackgroundSelf-citationTóthová, Z., Krill-Burger, J. M., Popova, K. D., Landers, C. C., Sievers, Q. L., Yudovich, D., Belizaire, R., Aster, J. C., Morgan, E. A., Tsherniak, A., et al. (2017) · Cell stem cell · doi:10.1016/j.stem.2017.07.015 · Cited by 96 · see citing works
“nucleases (TALENs), and RNA-guided nucleases (CRISPR/ Cas9), have been successfully employed to edit human HSPC (Buechele et al., 2015; Dever et al., 2016; Genovese et al., 2014b; Gundry et al., 2016; Mandal et al., 2014).”
BackgroundMethodLarge DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity
Zhang, X., Jeong, M., Huang, X., Wang, X. Q. D., Wang, X., Zhou, W., Shamim, M. S., Gore, H., Himadewi, P., Liu, Y., et al. (2020) · Molecular Cell · doi:10.1016/j.molcel.2020.04.018 · Cited by 91 · see citing works
Self-citationFreund, E. C., Lock, J. Y., Oh, J., Maculins, T., Delamarre, L., Bohlen, C. J., Haley, B., & Murthy, A. (2020) · The Journal of Experimental Medicine · doi:10.1084/jem.20191692 · Cited by 92 · see citing works
“A traditional method of differentiating BM cells into DCs involves supplementation with GM-CSF (Cornel et al., 2018; Gundry et al., 2016).”
MethodRoman‐Rodriguez, F. J., Ugalde, L., Alvarez, L. A., Díez, B., Ramı́rez, M. J., Risueño, C., Cortón, M., Bogliolo, M., Bernal, S., March, F., et al. (2019) · Cell stem cell · doi:10.1016/j.stem.2019.08.016 · Cited by 87 · see citing works
Pastore, N., Vainshtein, A., Herz, N. J., Huynh, T., Brunetti, L., Klisch, T. J., Mutarelli, M., Annunziata, P., Kinouchi, K., Brunetti‐Pierri, N., et al. (2019) · The EMBO Journal · doi:10.15252/embj.2018101347 · Cited by 83 · see citing works
“…Western blot analysis, cells were treated with 200 nM bafilomycin for 4 h. Generation of Bmal 1 /Clock KO Hepa 1 - 6 through CRISPR-Cas 9 Two sgRNAs targeting adjacent regions of Bmal1 exon 4 and Clock exon 17 were designed and synthetized according to Gundry et al (2016) with minimal modification.”
Self-citationGuide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells
Ting, P. Y., Parker, A. E., Lee, J. S., Trussell, C., Sharif, O., Luna, F., Federe, G., Barnes, S. W., Walker, J. R., Vance, J., et al. (2018) · Nature Methods · doi:10.1038/s41592-018-0149-1 · Cited by 80 · see citing works
Reimer, J., Knöß, S., Labuhn, M., Charpentier, E., Göhring, G., Schlegelberger, B., Klusmann, J., & Heckl, D. (2017) · Haematologica · doi:10.3324/haematol.2017.164046 · Cited by 75 · see citing works
Khosravi, M. A., Abbasalipour, M., Concordet, J., Berg, J. V., Zeinali, S., Arashkia, A., Azadmanesh, K., Buch, T., & Karimipoor, M. (2019) · European Journal of Pharmacology · doi:10.1016/j.ejphar.2019.04.042 · Cited by 71 · see citing works
NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus
Ando, K., Parsons, M. J., Shah, R. B., Charendoff, C. I., Paris, S. L., Liu, P. H., Fassio, S. R., Rohrman, B. A., Thompson, R. H., Oberst, A., et al. (2017) · The Journal of Cell Biology · doi:10.1083/jcb.201608095 · Cited by 68 · see citing works
“C2 Pro-BiFC cells using an adaptation of the protocol described in Gundry et al. (2016).”
Yen, J. S., Fiorino, M., Liu, Y., Paula, S., Clarkson, S., Quinn, L., Tschantz, W. R., Klock, H. E., Guo, N., Russ, C., et al. (2018) · Scientific Reports · doi:10.1038/s41598-018-34601-6 · Cited by 65 · see citing works
Dever, D. P., & Porteus, M. H. (2017) · Current Opinion in Hematology · doi:10.1097/moh.0000000000000385 · Cited by 62 · see citing works
Rinke, J., Chase, A., Cross, N. C. P., Hochhaus, A., & Ernst, T. (2020) · Cells · doi:10.3390/cells9071639 · Cited by 66 · see citing works
“Particularly promising are recent approaches, which focus on method optimization of CRISPR/Cas9 genome engineering on primary human HSCs and HPCs, followed by transplantation into immune deficient mice to generate models of clonal hematopoiesis and neoplasia [72,73].”
MethodWagner, D., Koehl, U., Chmielewski, M., Scheid, C., & Stripecke, R. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.865424 · Cited by 53 · see citing works
“Most published protocols use electroporation as means to introduce the RNP into the cell with minimal toxicity to T cells (56, 57) (58).”
MethodSelf-citationTovy, A., Spiro, A., McCarthy, R. L., Shipony, Z., Aylon, Y., Allton, K. L., Ainbinder, E., Furth, N., Tanay, A., Barton, M. C., et al. (2017) · Genes & Development · doi:10.1101/gad.299198.117 · Cited by 64 · see citing works
“Deletion of p53 by CRISPR/Cas9 was done as described (Gundry et al. 2016).”
Non-viral strategies for delivering genome editing enzymes
Li, J., Røise, J. J., He, M., Das, R., & Murthy, N. (2020) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2020.09.004 · Cited by 63 · see citing works
Synapse-tuned CARs enhance immune cell anti-tumor activity
Chockley, P. J., Ibañez-Vega, J., Krenciute, G., Talbot, L. J., & Gottschalk, S. M. (2023) · Nature Biotechnology · doi:10.1038/s41587-022-01650-2 · Cited by 64 · see citing works
Sommerkamp, P., Altamura, S., Renders, S., Narr, A., Ladel, L., Zeisberger, P., Eiben, P. L., Fawaz, M., Rieger, M., Cabezas-Wallscheid, N., et al. (2020) · Cell Stem Cell · doi:10.1016/j.stem.2020.03.003 · Cited by 57 · see citing works
Dai, Z., Mu, W., Zhao, Y., Jia, X., Liu, J., Wei, Q., Tan, T., & Zhou, J. (2021) · Molecular Therapy · doi:10.1016/j.ymthe.2021.07.001 · Cited by 62 · see citing works
“After 1 day of activation, the CD5 gene was genomically disrupted in primary human T cells using a Celetrix electroporation system (Celetrix, Manassas, VA, USA) following the manufacturer’s instructions.(48) Then, CD5KO-T cells were transduced with lentivirus at a multiplicity of infection of 2–5 1 day later.”
MethodShen, Y., Cohen, J. L., Nicoloro, S. M. C., Kelly, M., Yenilmez, B., Henriques, F. D. S., Tsagkaraki, E., Edwards, Y. J. K., Hu, X., Friedline, R. H., et al. (2018) · Journal of Biological Chemistry · doi:10.1074/jbc.ra118.004554 · Cited by 61 · see citing works
Amini, L., Wagner, D. L., Rössler, U., Zarrinrad, G., Wagner, L. F., Vollmer, T., Wendering, D. J., Kornak, U., Volk, H., Reinke, P., et al. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.09.011 · Cited by 56 · see citing works
“Efficient gene editing was achieved by transfection with ribonucleoprotein (RNP) complexes consisting of CRISPR-associated protein 9 (Cas9) and respective single guide RNA (sgRNA).(30) We implemented a RNP nucleofection step in our existing clinical protocol for generation of antiviral TCPs.”
MethodSelf-citationNuclear NAD + homeostasis governed by NMNAT1 prevents apoptosis of acute myeloid leukemia stem cells
Shi, X. E., Jiang, Y., Kitano, A., Hu, T., Murdaugh, R. L., Li, Y., Hoegenauer, K. A., Chen, R., Takahashi, K., & Nakada, D. (2021) · Science Advances · doi:10.1126/sciadv.abf3895 · Cited by 55 · see citing works
“12 of 15 introduced the Cas9-sgRNA complex to murine AML cells as previously described (38, 53).”
MethodSelf-citationWASP integrates substrate topology and cell polarity to guide neutrophil migration
Brunetti, R. M., Kockelkoren, G., Raghavan, P., Bell, G. R. R., Britain, D., Puri, N., Collins, S. R., Leonetti, M. D., Stamou, D. G., & Weiner, O. D. (2021) · The Journal of Cell Biology · doi:10.1083/jcb.202104046 · Cited by 54 · see citing works
Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer
Zeng, W., Zhang, R., Huang, P., Chen, M., Chen, H., Zeng, X., Liu, J., Zhang, J., Huang, D., & Lao, L. (2024) · Cancer Research · doi:10.1158/0008-5472.can-24-1941 · Cited by 54 · see citing works
“CRISPR-mediated gene knockout was performed as previously reported (23).”
Glia-specific enhancers and chromatin structure regulate NFIA expression and glioma tumorigenesis
Glasgow, S. M., Carlson, J. C., Zhu, W., Chaboub, L. S., Kang, P., Lee, H. K., Clovis, Y. M., Lozzi, B., McEvilly, R. J., Rosenfeld, M. G., et al. (2017) · Nature Neuroscience · doi:10.1038/nn.4638 · Cited by 52 · see citing works
TFEB regulates murine liver cell fate during development and regeneration
Pastore, N., Huynh, T., Herz, N. J., Calcagnì, A., Klisch, T. J., Brunetti, L., Kim, K. H., Giorgi, M. D., Hurley, A. E., Carissimo, A., et al. (2020) · Nature Communications · doi:10.1038/s41467-020-16300-x · Cited by 51 · see citing works
Self-citationRice, S., Jackson, T., Crump, N. T., Fordham, N., Elliott, N., O’Byrne, S., Fanego, M. D. M. L., Addy, D., Crabb, T., Dryden, C., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-27270-z · Cited by 52 · see citing works
Tran, N. T., Danner, E., Li, X., Graf, R., Lebedin, M., de la Rosa, K., Kühn, R., Rajewsky, K., & Chu, V. T. (2022) · Science Advances · doi:10.1126/sciadv.abm9106 · Cited by 49 · see citing works
Regulatory CAR-T cells in autoimmune diseases: Progress and current challenges
Riët, T., & Chmielewski, M. M. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.934343 · Cited by 54 · see citing works
“- To stabilize the in vivo suppressive function of Treg by i) epigenetic FOXP3 gene modification (66–69), ii) CRISPR/Cas9 mediated knocking-out of switch-point-receptors whose activation leads to loss of Treg function or even to the switch to effector cells (70, 71), or iii) mitochondrial modification (72– 74) iv) exp…”
BackgroundTargeting Acute Myeloid Leukemia Stem Cells through Perturbation of Mitochondrial Calcium
Sheth, A. I., Engel, K. L., Tolison, H., Althoff, M. J., Amaya, M. L., Krug, A. E., Young, T. N., Pei, S., Patel, S. B., Minhajuddin, M., et al. (2024) · Cancer Discovery · doi:10.1158/2159-8290.cd-23-1145 · Cited by 51 · see citing works
Gutiérrez-Guerrero, A., Abrey-Recalde, M. J., Mangeot, P. E., Costa, C., Bernadin, O., Perian, S., Fusil, F., Froment, G., Martinez-Turtos, A., Krug, A., et al. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.604371 · Cited by 51 · see citing works
“Gundry et al. (2016) evaluated the impact of Cas9/gRNA RNP electroporation into CD34+ cells.”
BackgroundGene Therapy in a Patient with Sickle Cell Disease
Badat, M., & Davies, J. (2017) · New England Journal of Medicine · doi:10.1056/nejmc1704009 · Cited by 54 · see citing works
Sweeney, C. L., Pavel-Dinu, M., Choi, U., Brault, J., Liu, T., Koontz, S. M., Li, L., Theobald, N., Lee, J., Bello, E. A., et al. (2021) · Gene Therapy · doi:10.1038/s41434-021-00251-z · Cited by 51 · see citing works
Cruz, L. J., van Dijk, T., Vepris, O., Li, T. M. W. Y., Schomann, T., Baldazzi, F., Kurita, R., Nakamura, Y., Grosveld, F. G., Philipsen, S., et al. (2020) · Biomaterials · doi:10.1016/j.biomaterials.2020.120580 · Cited by 50 · see citing works
“HSPC CRISPR-editing approaches utilize electroporation to deliver gRNA and Cas9 directly into target cells [14,22,28].”
BackgroundMethodFunctional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells
Wagenblast, E., Azkanaz, M., Smith, S. A., Shakib, L., McLeod, J. L., Krivdova, G., Araújo, J., Shultz, L. D., Gan, O. I., Dick, J. E., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-12726-0 · Cited by 49 · see citing works
A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing
Sinha, S., Barbosa, K., Cheng, K., Leiserson, M. D. M., Jain, P., Deshpande, A. J., Wilson, D. M., Ryan, B. M., Luo, J., Ronai, Z. A., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-26788-6 · Cited by 54 · see citing works
ERα promotes murine hematopoietic regeneration through the Ire1α-mediated unfolded protein response
Chapple, R. H., Hu, T., Tseng, Y.-J., Liu, L., Kitano, A., Luu, V., Hoegenauer, K. A., Iwawaki, T., Li, Q., & Nakada, D. (2018) · eLife · doi:10.7554/elife.31159 · Cited by 51 · see citing works
“Genome editing was performed essentially as described previously (Gundry et al., 2016).”
MethodSelf-citationRecent advances in genome editing of stem cells for drug discovery and therapeutic application
Lee, J., Bayarsaikhan, D., Bayarsaikhan, G., Kim, J., Schwarzbach, E., & Lee, B. (2020) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2020.107501 · Cited by 47 · see citing works
Noncanonical Roles of Caspase-4 and Caspase-5 in Heme-Driven IL-1β Release and Cell Death
Bolívar, B. E., Brown-Suedel, A. N., Rohrman, B. A., Charendoff, C. I., Yazdani, V., Belcher, J. D., Vercellotti, G. M., Flanagan, J. M., & Bouchier‐Hayes, L. (2021) · The Journal of Immunology · doi:10.4049/jimmunol.2000226 · Cited by 47 · see citing works
Heterozygous variants of CLPB are a cause of severe congenital neutropenia
Warren, J. T., Cupo, R. R., Wattanasirakul, P., Spencer, D. H., Locke, A. E., Makaryan, V., Bolyard, A. A., Kelley, M. L., Kingston, N. L., Shorter, J., et al. (2021) · Blood · doi:10.1182/blood.2021010762 · Cited by 47 · see citing works
Jiang, Y., Hu, T., Wang, T., Shi, X.-G., Kitano, A., Eagle, K., Hoegenauer, K. A., Konopleva, M., Lin, C. Y., Young, N. L., et al. (2019) · Blood · doi:10.1182/blood.2019001076 · Cited by 36 · see citing works
Self-citationTran, N. T., Graf, R., Wulf-Goldenberg, A., Stecklum, M., Strauß, G., Kühn, R., Kocks, C., Rajewsky, K., & Chu, V. T. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.08.004 · Cited by 46 · see citing works
LKB1/ STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms
Marinaccio, C., Suraneni, P. K., Celik, H., Volk, A. G., Wen, Q. J., Ling, T., Bulic, M., Lasho, T. L., Koche, R. P., Famulare, C. A., et al. (2021) · Cancer Discovery · doi:10.1158/2159-8290.cd-20-1353 · Cited by 46 · see citing works
García‐Tuñón, I., Hernández‐Sánchez, M., Ordóñez, J. L., Alonso-Pérez, V., Quijada‐Álamo, M., Benito, R., Guerrero, C., Hernández‐Rivas, J. M., & Sánchez‐Martín, M. (2017) · Oncotarget · doi:10.18632/oncotarget.15215 · Cited by 41 · see citing works
CRISPR to fix bad blood: a new tool in basic and clinical hematology
González-Romero, E., Martínez-Valiente, C., García-Ruiz, C., Vázquez‐Manrique, R. P., Cervera, J., & Sanjuán-Pla, A. (2019) · Haematologica · doi:10.3324/haematol.2018.211359 · Cited by 40 · see citing works
CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells
Kamali, E., Rahbarizadeh, F., Hojati, Z., & Frödin, M. (2021) · BMC Biotechnology · doi:10.1186/s12896-020-00665-4 · Cited by 40 · see citing works
“they are appearing very efficient and do not pose the toxicity associated with plasmids [39, 43, 44].”
BackgroundLee, B., Lozano, R. J., & Dunbar, C. E. (2021) · Molecular Therapy · doi:10.1016/j.ymthe.2021.09.001 · Cited by 38 · see citing works
Prolonged XPO1 inhibition is essential for optimal antileukemic activity in NPM1-mutated AML
Pianigiani, G., Gagliardi, A., Mezzasoma, F., Rocchio, F., Tini, V., Bigerna, B., Sportoletti, P., Caruso, S., Marra, A., Peruzzi, S., et al. (2022) · Blood Advances · doi:10.1182/bloodadvances.2022007563 · Cited by 39 · see citing works
“The cells were transfected with Cas9/ sgRNA ribonucleoproteins using the Neon Transfection System (Thermo Fisher Scientific) as previously described.(18) Loss of p53 was verified by Sanger sequencing and immunoblot analysis using mouse anti-p53 andmouse anti–β-actin antibodies.”
MethodSelf-citationMyoediting: Toward Prevention of Muscular Dystrophy by Therapeutic Genome Editing
Zhang, Y., Long, C., Bassel‐Duby, R., & Olson, E. N. (2018) · Physiological Reviews · doi:10.1152/physrev.00046.2017 · Cited by 40 · see citing works
“CRISPR/Cas-mediated ex vivo editing has been shown to be effective in the hematopoietic system (88, 125, 137, 345, 423).”
BackgroundMa, Y., Han, X., Bustamante, O. Q., de Castro, R. B., Zhang, K., Zhang, P., Li, Y., Liu, Z., Liu, X., Mauro Ferrari, B., et al. (2017) · Integrative Biology · doi:10.1039/c7ib00060j · Cited by 37 · see citing works
Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13–induced Mucostasis
Koh, K. D., Siddiqui, S., Cheng, D., Bonser, L. R., Sun, D. I., Zlock, L. T., Finkbeiner, W. E., Woodruff, P. G., & Erle, D. J. (2019) · American Journal of Respiratory Cell and Molecular Biology · doi:10.1165/rcmb.2019-0266oc · Cited by 38 · see citing works
An oncogenic enhancer encodes selective selenium dependency in AML
Eagle, K., Jiang, Y., Shi, X., Li, M., Obholzer, N., Hu, T., Perez, M. W., Koren, J., Kitano, A., Yi, J. S., et al. (2022) · Cell Stem Cell · doi:10.1016/j.stem.2022.01.003 · Cited by 21 · see citing works
Self-citationGene therapy and genome editing for primary immunodeficiency diseases
Zhang, Z., Thrasher, A. J., & Zhang, F. (2019) · Genes & Diseases · doi:10.1016/j.gendis.2019.07.007 · Cited by 37 · see citing works
Secker, K.-A., Keppeler, H., Duerr‐Stoerzer, S., Schmid, H., Schneidawind, D., Hentrich, T., Schulze-Hentrich, J. M., Mankel, B., Fend, F., & Schneidawind, C. (2019) · Oncogene · doi:10.1038/s41388-019-0937-9 · Cited by 37 · see citing works
Bianco, J., Rosa, R. G., Congrains, A., Joazeiro, P. P., Waldman, S. D., Weber, J. F., & Saad, S. T. O. (2019) · Biomaterials Science · doi:10.1039/c8bm01503a · Cited by 35 · see citing works
Celik, H., Krug, E., Zhang, C. R., Han, W., Issa, N., Koh, W. K., Bjeije, H., Kukhar, O., Allen, M., Li, T., et al. (2021) · Cancer Discovery · doi:10.1158/2159-8290.cd-20-1652 · Cited by 34 · see citing works
HSF1 is a driver of leukemia stem cell self-renewal in acute myeloid leukemia
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Glaucoma, Stem Cells, and Gene Therapy: Where Are We Now?
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“ing, and need specialized equipment, protocols, and expertise (66, 67).”
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A Costimulatory CAR Improves TCR-based Cancer Immunotherapy
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“A CD19-knockout (KO) BV173 line was generated by CRISPR/Cas9 as previously described (17).”
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“Furthermore, the short half-life of RNP minimizes off-target cleavage and limits HSPC toxicity: RNP-treated HSPCs did not show reduction in methylcellulose clonogenic capacity or cell viability (Gundry et al., 2016).”
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PRDM16s transforms megakaryocyte-erythroid progenitors to acute myeloid leukemia-initiating cells.
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- Authors
- Gundry, M. C., Brunetti, L., Lin, A., Mayle, A. E., Kitano, A., Wagner, D., Hsu, J. I., Hoegenauer, K. A., Rooney, C. M., Goodell, M. A., & Nakada, D.
- Affiliations
- Baylor College of Medicine (US)
- Houston Methodist (US)
- Corresponding author
- Goodell, Margaret A.
- Nakada, Daisuke
- ORCID
- Venue
- Cell Reports
- Publisher
- Elsevier BV
- Published
- 2016-10
- Volume
- 17
- Issue
- 5
- Pages
- 1453-1461
- ISSN
- 2211-1247 (ISSN-L)
- PMC
- PMC5087995
- OpenAlex
- W2535622376
- Cited by
- 265
- References
- 36
- Citations by year
- 2026: 12 · 2025: 10 · 2024: 19 · 2023: 24 · 2022: 31 · 2021: 34 · 2020: 44 · 2019: 35 · 2018: 32 · 2017: 22 · 2016: 2
- FWCI
- 15.76
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cell Reports (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Università Politecnica delle Marche (Università Politecnica delle Marche) (submittedVersion)
- Refubium (Universitätsbibliothek der Freien Universität Berlin) (submittedVersion, cc-by-nc-nd)
- Funders
- Cancer Prevention and Research Institute of Texas (RP16028, RP140001, R1201)
- NIH (DK092883, CA183252, CA125123, P50CA126752, CA193235, DK107413)
- National Center for Research Resources grant (S10RR024574)
- National Institute of Allergy and Infectious Diseases (AI036211)
- National Cancer Institute (P30CA125123)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, genome editing, hematopoietic stem cells, hematopoietic progenitor cells, ribonucleoprotein delivery, knockout mice, human hematopoietic cells, transfection efficiency, off-target effects
- MeSH terms
- Gene Editing, Animals, DNA Repair, Hematopoietic Stem Cells, Humans, Gene Deletion, Leukocyte Common Antigens, Mice, Gene Knockout Techniques, CRISPR-Cas Systems
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