The DOI matches its Crossref record; 56 of the 57 references it cites were found in a registry or an index.
CRISPR/Cas9 genome editing in human hematopoietic stem cells.
Open accessAbstract
Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.
Overview
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The cited source says what the claim attributes to it.
In the citing paper
“The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease system, a bacterial immune system that can cleave bacteriophage or plasmid DNA, enables programmable targeting of insertions or deletions (indels) at a specific genomic DNA site.28,29”
From CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia (2021) · cites this paper as [28]
Experimental — accuracy not yet measured
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Citations
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339
Genome engineering using the CRISPR-Cas9 system
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013) · Nature Protocols · doi:10.1038/nprot.2013.143 · Cited by 11,133
In vivo genome editing using Staphylococcus aureus Cas9
Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., et al. (2015) · Nature · doi:10.1038/nature14299 · Cited by 2,687
Trypan Blue Exclusion Test of Cell Viability
Strober, W. (2015) · Current Protocols in Immunology · doi:10.1002/0471142735.ima03bs111 · Cited by 2,631
Engineered CRISPR-Cas9 nucleases with altered PAM specificities
Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., Gonzales, A. P. W., Li, Z., Peterson, R. T., Yeh, J.-R. J., et al. (2015) · Nature · doi:10.1038/nature14592 · Cited by 1,624
Humanized mice in translational biomedical research
Shultz, L. D., Ishikawa, F., & Greiner, D. L. (2007) · Nature reviews. Immunology · doi:10.1038/nri2017 · Cited by 1,393
Trypan Blue Exclusion Test of Cell Viability
Strober, W. (1997) · Current Protocols in Immunology · doi:10.1002/0471142735.ima03bs21 · Cited by 1,349
Therapeutic genome editing: prospects and challenges
Cox, D. B. T., Platt, R. J., & Zhang, F. (2015) · Nature Medicine · doi:10.1038/nm.3793 · Cited by 1,336
Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells
Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3290 · Cited by 1,090
Self-citationIsolation of a candidate human hematopoietic stem-cell population.
Baum, C. M., Weissman, I. L., Tsukamoto, A. S., Buckle, A., & Péault, B. (1992) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.89.7.2804 · Cited by 1,084
Humanized mice in translational biomedical research
Shultz, L. (2007) · The Mouseion at the JAXlibrary (Jackson Laboratory) · Cited by 1,036
Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment
Notta, F., Doulatov, S., Laurenti, E., Poeppl, A., Jurisica, I., & Dick, J. E. (2011) · Science · doi:10.1126/science.1201219 · Cited by 919
Hematopoiesis: a human perspective
Doulatov, S., Notta, F., Laurenti, E., & Dick, J. E. (2012) · Cell stem cell · doi:10.1016/j.stem.2012.01.006 · Cited by 895
CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells
Dever, D. P., Bak, R. O., Reinisch, A., Camarena, J., Washington, G., Nicolas, C. E., Pavel-Dinu, M., Saxena, N., Wilkens, A. B., Mantri, S., et al. (2016) · Nature · doi:10.1038/nature20134 · Cited by 864
Self-citationChimeric nucleases stimulate gene targeting in human cells
Porteus, M. H., & Baltimore, D. (2003) · Science · doi:10.1126/science.1078395 · Cited by 741
Self-citationGeneration of knock-in primary human T cells using Cas9 ribonucleoproteins
Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., Haliburton, G. E., Ye, C. J., Bluestone, J. A., Doudna, J. A., et al. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1512503112 · Cited by 702
Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood
Majeti, R., Park, C. Y., & Weissman, I. L. (2007) · Cell stem cell · doi:10.1016/j.stem.2007.10.001 · Cited by 657
Systemic delivery of genes to striated muscles using adeno-associated viral vectors
Gregorevic, P., Blankinship, M. J., Allen, J. M., Crawford, R. W., Meuse, L., Miller, D. G., Russell, D. W., & Chamberlain, J. S. (2004) · Nature Medicine · doi:10.1038/nm1085 · Cited by 620
Production and characterization of adeno-associated viral vectors
Grieger, J. C., Choi, V. W., & Samulski, R. J. (2006) · Nature Protocols · doi:10.1038/nprot.2006.207 · Cited by 580
Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.
Rouet, P., Smih, F., & Jasin, M. (1994) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.91.13.6064 · Cited by 571
Targeted genome editing in human repopulating haematopoietic stem cells
Genovese, P., Schiroli, G., Escobar, G., Di Tomaso, T., Firrito, C., Calabria, A., Moi, D., Mazzieri, R., Bonini, C., Holmes, M. C., et al. (2014) · Nature · doi:10.1038/nature13420 · Cited by 555
Aurnhammer, C., Haase, M., Müther, N., Mück-Häusl, M., Rauschhuber, C., Huber, I., Nitschko, H., Busch, U., Sing, A., Ehrhardt, A., et al. (2012) · Human Gene Therapy Methods · doi:10.1089/hgtb.2011.034 · Cited by 494
DeWitt, M. A., Magis, W., Bray, N. L., Wang, T., Berman, J. R., Urbinati, F., Heo, S.-J., Mitros, T., Muñoz, D. P., Boffelli, D., et al. (2016) · Science Translational Medicine · doi:10.1126/scitranslmed.aaf9336 · Cited by 477
Ex vivo gene transfer and correction for cell-based therapies
Naldini, L. (2011) · Nature Reviews Genetics · doi:10.1038/nrg2985 · Cited by 395
Expansion of human SCID-repopulating cells under hypoxic conditions
Danet, G. H., Pan, Y., Luongo, J. L., Bonnet, D. A., & Simon, M. C. (2003) · Journal of Clinical Investigation · doi:10.1172/jci17669 · Cited by 358
Collaboration and competition between DNA double‐strand break repair pathways
Kass, E. M., & Jasin, M. (2010) · FEBS Letters · doi:10.1016/j.febslet.2010.07.057 · Cited by 326
Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells
Hoban, M. D., Cost, G. J., Mendel, M. C., Romero, Z., Kaufman, M. L., Joglekar, A. V., Ho, M., Lumaquin, D., Gray, D., Lill, G. R., et al. (2015) · Blood · doi:10.1182/blood-2014-12-615948 · Cited by 320
Human gene targeting by viral vectors
Russell, D. W., & Hirata, R. K. (1998) · Nature Genetics · doi:10.1038/ng0498-325 · Cited by 316
Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells
Elliott, B., Richardson, C. A., Winderbaum, J., Nickoloff, J. A., & Jasin, M. (1998) · Molecular and Cellular Biology · doi:10.1128/mcb.18.1.93 · Cited by 305
Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
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., et al. (2016) · Cell Reports · doi:10.1016/j.celrep.2016.09.092 · Cited by 265
Promoterless gene targeting without nucleases ameliorates haemophilia B in mice
Barzel, A., Paulk, N. K., Shi, Y., Huang, Y., Chu, K. C., Zhang, F., Valdmanis, P. N., Spector, L. P., Porteus, M. H., Gaensler, K. M. L., et al. (2014) · Nature · doi:10.1038/nature13864 · Cited by 264
Self-citationDe Ravin, S. S., Li, L., Wu, X., Choi, U., Allen, C., Koontz, S., Lee, J., Theobald-Whiting, N., Chu, J., Garofalo, M., et al. (2017) · Science Translational Medicine · doi:10.1126/scitranslmed.aah3480 · Cited by 258
Reinisch, A., Thomas, D., Corces, M. R., Zhang, X., Gratzinger, D. A., Hong, W., Schallmoser, K., Strunk, D., & Majeti, R. (2016) · Nature Medicine · doi:10.1038/nm.4103 · Cited by 229
Sather, B. D., Romano Ibarra, G. S., Sommer, K., Curinga, G., Hale, M., Khan, I. F., Singh, S., Song, Y., Gwiazda, K., Sahni, J., et al. (2015) · Science Translational Medicine · doi:10.1126/scitranslmed.aac5530 · Cited by 223
Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases
Osborn, M. J., Webber, B. R., Knipping, F., Lonetree, C.-L., Tennis, N., DeFeo, A. P., McElroy, A. N., Starker, C. G., Lee, C., Merkel, S., et al. (2016) · Molecular Therapy · doi:10.1038/mt.2015.197 · Cited by 201
De Ravin, S. S., Reik, A., Liu, P.-Q., Li, L., Wu, X., Su, L., Raley, C., Theobald, N., Choi, U., Song, A. H., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3513 · Cited by 195
AAV-mediated gene targeting methods for human cells
Khan, I., Hirata, R. K., & Russell, D. W. (2011) · Nature Protocols · doi:10.1038/nprot.2011.301 · Cited by 188
Psaila, B., Barkas, N., Iskander, D., Roy, A., Anderson, S. M., Ashley, N., Caputo, V. S., Lichtenberg, J., Loaiza, S., Bodine, D. M., et al. (2016) · Genome biology · doi:10.1186/s13059-016-0939-7 · Cited by 165
Notta, F., Doulatov, S. R., & Dick, J. E. (2010) · Blood · doi:10.1182/blood-2009-10-249326 · Cited by 155
An Efficient Low Cost Method for Gene Transfer to T Lymphocytes
Chicaybam, L., Sodré, A. L., Curzio, B. A., & Bonamino, M. (2013) · PLoS ONE · doi:10.1371/journal.pone.0060298 · Cited by 143
Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome
Hubbard, N., Hagin, D., Sommer, K., Song, Y., Khan, I., Clough, C. A., Ochs, H. D., Rawlings, D. J., Scharenberg, A. M., & Torgerson, T. R. (2016) · Blood · doi:10.1182/blood-2015-11-683235 · Cited by 140
Design and Packaging of Adeno-Associated Virus Gene Targeting Vectors
Hirata, R. K., & Russell, D. W. (2000) · Journal of Virology · doi:10.1128/jvi.74.10.4612-4620.2000 · Cited by 133
Human Gene Targeting by Adeno-Associated Virus Vectors Is Enhanced by DNA Double-Strand Breaks
Miller, D. G., Petek, L. M., & Russell, D. (2003) · Molecular and Cellular Biology · doi:10.1128/mcb.23.10.3550-3557.2003 · Cited by 132
Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material
Lock, M., McGorray, S., Auricchio, A., Ayuso, E., Beecham, E. J., Blouin-Tavel, V., Bosch, F., Bose, M., Byrne, B. J., Caton, T., et al. (2010) · Human Gene Therapy · doi:10.1089/hum.2009.223 · Cited by 131
Wang, J., DeClercq, J. J., Hayward, S. B., Li, P. W.-L., Shivak, D. A., Gregory, P. D., Lee, G., & Holmes, M. C. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkv1121 · Cited by 128
Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing
Hendel, A., Kildebeck, E., Fine, E. J., Clark, J. T., Punjya, N., Sebastiano, V., Bao, G., & Porteus, M. H. (2014) · Cell Reports · doi:10.1016/j.celrep.2014.02.040 · Cited by 124
Self-citationCRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors
Bak, R. O., & Porteus, M. H. (2017) · Cell Reports · doi:10.1016/j.celrep.2017.06.064 · Cited by 124
Self-citationBak, R. O., Dever, D. P., Reinisch, A., Cruz Hernandez, D., Majeti, R., & Porteus, M. H. (2017) · eLife · doi:10.7554/elife.27873 · Cited by 122
Self-citationGenetic knockouts and knockins in human somatic cells
Rago, C., Vogelstein, B., & Bunz, F. (2007) · Nature Protocols · doi:10.1038/nprot.2007.408 · Cited by 120
Genome Editing: A New Approach to Human Therapeutics
Porteus, M. H. (2015) · The Annual Review of Pharmacology and Toxicology · doi:10.1146/annurev-pharmtox-010814-124454 · Cited by 118
Self-citationKoller, M. R., Bender, J., Papoutsakis, E., & Miller, W. (1992) · Blood · doi:10.1182/blood.v80.2.403.403 · Cited by 83
Towards a new era in medicine: therapeutic genome editing
Porteus, M. H. (2015) · Genome Biology · doi:10.1186/s13059-015-0859-y · Cited by 69
Self-citationPark, C. Y., Majeti, R., & Weissman, I. L. (2008) · Nature Protocols · doi:10.1038/nprot.2008.194 · Cited by 56
Song, L., Li, X., Jayandharan, G. R., Wang, Y., Aslanidi, G., Ling, C., Li, Z., Gao, G., Yöder, M. C., Ling, C., et al. (2013) · PLoS ONE · doi:10.1371/journal.pone.0058757 · Cited by 55
Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene
Hollywood, J. A., Lee, C. M., Scallan, M. F., & Harrison, P. T. (2016) · Scientific Reports · doi:10.1038/srep32230 · Cited by 34
Flagged first, then by citations
1 of the 57 references couldn’t be matched to a known paper.
Cited by
Sorted by citations
CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia
Frangoul, H., Altshuler, D., Cappellini, M. D., Chen, Y.-S., Domm, J., Eustace, B. K., Foell, J., de la Fuente, J., Grupp, S., Handgretinger, R., et al. (2021) · New England Journal of Medicine · doi:10.1056/nejmoa2031054 · Cited by 1,962
CRISPR-Cas guides the future of genetic engineering
Knott, G. J., & Doudna, J. A. (2018) · Science · doi:10.1126/science.aat5011 · Cited by 1,410
Tuning the Antigen Density Requirement for CAR T-cell Activity
Majzner, R. G., Rietberg, S. P., Sotillo, E., Dong, R., Vachharajani, V. T., Labanieh, L., Myklebust, J. H., Kadapakkam, M., Weber, E. W., Tousley, A. M., et al. (2020) · Cancer Discovery · doi:10.1158/2159-8290.cd-19-0945 · Cited by 596
Next-generation regulatory T cell therapy
Ferreira, L. M. R., Müller, Y. D., Bluestone, J. A., & Tang, Q. (2019) · Nature Reviews Drug Discovery · doi:10.1038/s41573-019-0041-4 · Cited by 491
Vaidyanathan, S., Azizian, K. T., Haque, A. K. M. A., Henderson, J. M., Hendel, A., Shore, S., Antony, J. S., Hogrefe, R. I., Kormann, M. S. D., Porteus, M. H., et al. (2018) · Molecular Therapy Nucleic Acids · doi:10.1016/j.omtn.2018.06.010 · Cited by 262
“3 mg Cas9 mRNA and 2 mg MS-sgRNA were used for nucleofections.(6,69) Cas9 mRNA and RNP were then mixed with 100,000 CD34 cells (20 mL of cell suspension) and transferred to a 16-well nucleofection strip (Lonza, MD, USA).”
MethodSelf-citation
Metadata
- Authors
- Bak, R. O., Dever, D. P., & Porteus, M. H.
- Affiliations
- Stanford Medicine (US)
- Corresponding author
- Porteus, Matthew H
- Venue
- Nature Protocols
- Published
- 2018-01-25
- Volume
- 13
- Issue
- 2
- Pages
- 358-376
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC5826598
- OpenAlex
- W2784423079
- Cited by
- 319
- References
- 57
- Citations by year
- 2026: 23 · 2025: 37 · 2024: 31 · 2023: 47 · 2022: 35 · 2021: 51 · 2020: 50 · 2019: 31 · 2018: 13
- FWCI
- 12.88
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (5r01ai120766-02)
- National Institute of Allergy and Infectious Diseases (R01-AI120766, R01AI097320)
- 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 › Hematology › Hematopoietic Stem Cell Transplantation
- Keywords
- CRISPR/Cas9 genome editing, hematopoietic stem cells, homologous recombination, AAV6 vector, Cas9 ribonucleoprotein, HSC engraftment, hematopoietic stem and progenitor cells, sickle cell disease, beta-thalassemia, primary immunodeficiency, gene therapy
- MeSH terms
- Gene Editing, Dependovirus, Animals, Flow Cytometry, Hematopoiesis, Hematopoietic Stem Cells, Humans, Genetic Therapy, Hematopoietic Stem Cell Transplantation, Gene Targeting, Polymorphism, Single Nucleotide, Mice, Homologous Recombination, CRISPR-Cas Systems
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
