The DOI matches its Crossref record; 53 of the 54 references it cites were found in a registry or an index.
Orthologous CRISPR–Cas9 enzymes for combinatorial genetic screens
Open accessAbstract
Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
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?
In the citing paper
“ECCITE-seq is readily applicable to any sgRNA library with the Cas9 S. pyogenes scaffold sequence and, by allowing direct capture of sgRNA molecules, overcomes documented problems of barcode swapping events observed with Perturb-seq that have the potential to confound single cell perturbation screens17–20.”
From Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells (2019) · cites this paper as [19]
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
Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., & Wang, X. (1997) · Cell · doi:10.1016/s0092-8674(00)80434-1 · Cited by 7,289
Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy
Farmer, H., McCabe, N., Lord, C. J., Tutt, A. N. J., Johnson, D. A., Richardson, T. B., Santarosa, M., Dillon, K. J., Hickson, I., Knights, C., et al. (2005) · Nature · doi:10.1038/nature03445 · Cited by 6,683
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3437 · Cited by 4,637
Self-citationMitochondrial membrane permeabilization in cell death
Kroemer, G., Galluzzi, L., & Brenner, C. (2007) · Physiological Reviews · doi:10.1152/physrev.00013.2006 · Cited by 3,598
An inhibitor of Bcl-2 family proteins induces regression of solid tumours
Oltersdorf, T., Elmore, S. W., Shoemaker, A. R., Armstrong, R. C., Augeri, D. J., Belli, B., Bruncko, M., Deckwerth, T. L., Dinges, J., Hajduk, P. J., et al. (2005) · Nature · doi:10.1038/nature03579 · Cited by 3,407
Discovery and saturation analysis of cancer genes across 21 tumour types
Lawrence, M. S., Stojanov, P., Mermel, C. H., Robinson, J. T., Garraway, L. A., Golub, T. R., Meyerson, M., Gabriel, S. B., Lander, E. S., & Getz, G. (2014) · Nature · doi:10.1038/nature12912 · Cited by 3,096
ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets
Souers, A. J., Leverson, J. D., Boghaert, E. R., Ackler, S. L., Catron, N. D., Chen, J., Dayton, B. D., Ding, H., Enschede, S. H., Fairbrother, W. J., et al. (2013) · Nature Medicine · doi:10.1038/nm.3048 · Cited by 3,058
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
Gilbert, L. A., Horlbeck, M. A., Adamson, B., Villalta, J. E., Chen, Y., Whitehead, E. H., Guimaraes, C., Panning, B., Ploegh, H. L., Bassik, M. C., et al. (2014) · Cell · doi:10.1016/j.cell.2014.09.029 · Cited by 2,728
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
Global Mapping of the Yeast Genetic Interaction Network
Tong, A. H. Y., Lesage, G., Bader, G. D., Ding, H., Xu, H., Xin, X., Young, J., Berriz, G. F., Brost, R. L., Chang, M., et al. (2004) · Science · doi:10.1126/science.1091317 · Cited by 2,089
COSMIC: somatic cancer genetics at high-resolution
Forbes, S. A., Beare, D., Boutselakis, H., Bamford, S., Bindal, N., Tate, J., Cole, C. G., Ward, S., Dawson, E., Ponting, L., et al. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkw1121 · Cited by 1,922
Highly efficient Cas9-mediated transcriptional programming
Chavez, A., Scheiman, J., Vora, S., Pruitt, B. W., Tuttle, M., P R Iyer, E., Lin, S., Kiani, S., Guzman, C. D., Wiegand, D. J., et al. (2015) · Nature Methods · doi:10.1038/nmeth.3312 · Cited by 1,622
Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation
Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B. L., Xavier, R. J., & Root, D. E. (2014) · Nature Biotechnology · doi:10.1038/nbt.3026 · Cited by 1,617
Self-citationHigh-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.
Hart, T., Chandrashekhar, M., Aregger, M., Steinhart, Z., Brown, K. R., MacLeod, G., Mis, M., Zimmermann, M., Fradet-Turcotte, A., Sun, S., et al. (2015) · Cell · doi:10.1016/j.cell.2015.11.015 · Cited by 1,571
van Delft, M. F., Wei, A. H., Mason, K. D., Vandenberg, C. J., Chen, L., Czabotar, P. E., Willis, S. N., Scott, C. L., Day, C. L., Cory, S., et al. (2006) · Cancer Cell · doi:10.1016/j.ccr.2006.08.027 · Cited by 1,271
A global genetic interaction network maps a wiring diagram of cellular function
Costanzo, M., VanderSluis, B., Koch, E. N., Baryshnikova, A., Pons, C., Tan, G., Wang, W., Usaj, M., Hanchard, J., Lee, S. D., et al. (2016) · Science · doi:10.1126/science.aaf1420 · Cited by 1,240
Adamson, B., Norman, T. M., Jost, M., Cho, M. Y., Nuñez, J. K., Chen, Y., Villalta, J. E., Gilbert, L. A., Horlbeck, M. A., Hein, M. Y., et al. (2016) · Cell · doi:10.1016/j.cell.2016.11.048 · Cited by 1,193
The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models
Kotschy, A., Szlavik, Z., Murray, J., Davidson, J., Maragno, A. L., Le Toumelin-Braizat, G., Chanrion, M., Kelly, G. L., Gong, J.-N., Moujalled, D. M., et al. (2016) · Nature · doi:10.1038/nature19830 · Cited by 1,047
Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array
Zetsche, B., Heidenreich, M., Mohanraju, P., Fedorova, I., Kneppers, J., DeGennaro, E. M., Winblad, N., Choudhury, S. R., Abudayyeh, O. O., Gootenberg, J. S., et al. (2017) · Nature Biotechnology · doi:10.1038/nbt.3737 · Cited by 926
Helleday, T. (2011) · Molecular Oncology · doi:10.1016/j.molonc.2011.07.001 · Cited by 898
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition
Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Topkar, V. V., Zheng, Z., & Joung, J. K. (2015) · Nature Biotechnology · doi:10.1038/nbt.3404 · Cited by 611
Leverson, J. D., Phillips, D. C., Mitten, M. J., Boghaert, E. R., Diaz, D., Tahir, S. K., Belmont, L. D., Nimmer, P., Xiao, Y., Ma, X. M., et al. (2015) · Science Translational Medicine · doi:10.1126/scitranslmed.aaa4642 · Cited by 541
Han, K., Jeng, E. E., Hess, G. T., Morgens, D. W., Li, A., & Bassik, M. C. (2017) · Nature Biotechnology · doi:10.1038/nbt.3834 · Cited by 498
Zhu, S., Li, W., Liu, J., Chen, C.-H., Liao, Q., Xu, P., Xu, H., Xiao, T., Cao, Z., Peng, J., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3715 · Cited by 427
Structure-guided design of a selective BCL-XL inhibitor
Lessène, G., Czabotar, P. E., Sleebs, B. E., Zobel, K., Lowes, K. N., Adams, J. M., Baell, J. B., Colman, P. M., Deshayes, K., Fairbrother, W. J., et al. (2013) · Nature Chemical Biology · doi:10.1038/nchembio.1246 · Cited by 393
Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review)
Nițulescu, G. M., Margina, D. M., Juzenas, P., Peng, Q., Olaru, O. T., Saloustros, E. S., Fenga, C., Spandidos, D. A., Libra, M., & Tsatsakis, A. M. (2015) · International Journal of Oncology · doi:10.3892/ijo.2015.3306 · Cited by 382
A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility
Bassik, M. C., Kampmann, M., Lebbink, R. J., Wang, S., Hein, M. Y., Poser, I., Weibezahn, J., Horlbeck, M. A., Chen, S., Mann, M., et al. (2013) · Cell · doi:10.1016/j.cell.2013.01.030 · Cited by 365
Combinatorial CRISPR–Cas9 screens for de novo mapping of genetic interactions
Shen, J. P., Zhao, D., Sasik, R., Luebeck, J., Birmingham, A., Bojorquez-Gomez, A., Licon, K., Klepper, K., Pekin, D., Beckett, A. N., et al. (2017) · Nature Methods · doi:10.1038/nmeth.4225 · Cited by 359 · cited 3×
“Two efforts to achieve combinatorial CRISPR screens employed orthologous U6 promoters, from mouse and human 7,8 , although another study found that multiple copies of the S. pyogenes tracrRNA sequence were likewise prone to recombination 11 .”
p53 Suppresses the Nrf2-dependent Transcription of Antioxidant Response Genes
Faraonio, R., Vergara, P., Marzo, D. D., Pierantoni, G. M., Napolitano, M., Russo, T., & Cimino, F. (2006) · Journal of Biological Chemistry · doi:10.1074/jbc.m605707200 · Cited by 351
Complex transcriptional modulation with orthogonal and inducible dCas9 regulators
Gao, Y., Xiong, X., Wong, S. S., Charles, E. J., Lim, W. A., & Qi, L. S. (2016) · Nature Methods · doi:10.1038/nmeth.4042 · Cited by 335
ERK1 and ERK2 Map Kinases: Specific Roles or Functional Redundancy?
Buscà, R., Pouysségur, J., & Lenormand, P. (2016) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2016.00053 · Cited by 303
Dummler, B. A., Tschopp, O., Hynx, D., Yang, Z., Dirnhofer, S., & Hemmings, B. A. (2006) · Molecular and Cellular Biology · doi:10.1128/mcb.00722-06 · Cited by 293
Friedland, A. E., Baral, R., Singhal, P., Loveluck, K., Shen, S., Sanchez, M. E., Marco, E., Gotta, G. M., Maeder, M. L., Kennedy, E. M., et al. (2015) · Genome biology · doi:10.1186/s13059-015-0817-8 · Cited by 285
Sarosiek, K. A., Chi, X., Bachman, J. A., Sims, J. J., Montero, J., Patel, L., Flanagan, A., Andrews, D. W., Sorger, P. K., & Letai, A. G. (2013) · Molecular Cell · doi:10.1016/j.molcel.2013.08.048 · Cited by 252
Cowling, V. H., & Downward, J. (2002) · Cell Death and Differentiation · doi:10.1038/sj.cdd.4401065 · Cited by 249
Lentiviral Vector Design for Multiple shRNA Expression and Durable HIV-1 Inhibition
ter Brake, O., Hooft, K. '., Liu, Y. P., Centlivre, M., von Eije, K. J., & Berkhout, B. (2008) · Molecular Therapy · doi:10.1038/sj.mt.6300382 · Cited by 241
Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM
Wong, A. S. L., Choi, G. C. G., Cui, C. H., Pregernig, G., Milani, P., Adam, M., Perli, S. D., Kazer, S. W., Gaillard, A., Hermann, M., et al. (2016) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1517883113 · Cited by 240
Hanzlíková, H., Gittens, W. H., Krejčíková, K., Zeng, Z., & Caldecott, K. W. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw1246 · Cited by 204
Mapping of signaling networks through synthetic genetic interaction analysis by RNAi
Horn, T., Sandmann, T., Fischer, B., Axelsson, E., Huber, W., & Boutros, M. (2011) · Nature Methods · doi:10.1038/nmeth.1581 · Cited by 193
Stordal, B., Timms, K. M., Farrelly, A. M., Gallagher, D., Busschots, S., Renaud, M., Thery, J., Williams, D., Potter, J., Tran, T., et al. (2013) · Molecular Oncology · doi:10.1016/j.molonc.2012.12.007 · Cited by 161
Feedback and redundancy in receptor tyrosine kinase signaling: relevance to cancer therapies
Sun, C., & Bernards, R. (2014) · Trends in Biochemical Sciences · doi:10.1016/j.tibs.2014.08.010 · Cited by 155
Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries
Vidigal, J. A., & Ventura, A. (2015) · Nature Communications · doi:10.1038/ncomms9083 · Cited by 147
A Network of Conserved Synthetic Lethal Interactions for Exploration of Precision Cancer Therapy
Srivas, R., Shen, J. P., Yang, C. C., Sun, S. M., Li, J., Gross, A. M., Jensen, J., Licon, K., Bojorquez-Gomez, A., Klepper, K., et al. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.06.022 · Cited by 143
Ma, S. B., Nguyen, T. N., Tan, I., Ninnis, R., Iyer, S., Stroud, D. A., Menard, M., Kluck, R. M., Ryan, M. T., & Dewson, G. (2014) · Cell Death & Differentiation · doi:10.1038/cdd.2014.119 · Cited by 138
Inactivating CUX1 mutations promote tumorigenesis
Wong, C. C., Martin, S., Rust, A. G., Rashid, M., Alifrangis, C., Alexandrov, L. B., Tiffen, J. C., Kober, C., Green, A. R., Massie, C. E., et al. (2013) · Nature Genetics · doi:10.1038/ng.2846 · Cited by 135
Rotem, A., Janzer, A., Izar, B., Ji, Z., Doench, J. G., Garraway, L. A., & Struhl, K. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1505979112 · Cited by 125
Self-citationUzgare, A. R., & Isaacs, J. T. (2004) · Cancer Research · doi:10.1158/0008-5472.can-04-0968 · Cited by 121
Antiapoptotic potency of Bcl-2 proteins primarily relies on their stability, not binding selectivity
Rooswinkel, R. W., van de Kooij, B., de Vries, E., Paauwe, M., Braster, R., Verheij, M., & Borst, J. G. (2014) · Blood · doi:10.1182/blood-2013-08-519470 · Cited by 110
Quantitative genetic-interaction mapping in mammalian cells
Roguev, A., Talbot, D., Negri, G. L., Shales, M., Cagney, G., Bandyopadhyay, S., Panning, B., & Krogan, N. J. (2013) · Nature Methods · doi:10.1038/nmeth.2398 · Cited by 105
Chaudhuri, L., Vincelette, N. D., Koh, B. D., Naylor, R. M., Flatten, K. S., Peterson, K. L., McNally, A., Gojo, I., Karp, J. E., Mesa, R. A., et al. (2013) · Haematologica · doi:10.3324/haematol.2013.093187 · Cited by 78
Miura, Y., Kataoka, H., Joh, T., Tada, T., Asai, K., Nakanishi, M., Okada, N., & Okada, H. (2004) · Microbiology and Immunology · doi:10.1111/j.1348-0421.2004.tb03491.x · Cited by 33
A comparison of multiple shRNA expression methods for combinatorial RNAi
Mcintyre, G. J., Arndt, A. J., Gillespie, K. M., Mak, W. M., & Fanning, G. C. (2011) · Genetic Vaccines and Therapy · doi:10.1186/1479-0556-9-9 · Cited by 20
A High-Throughput Strategy for Dissecting Mammalian Genetic Interactions
Stockman, V. B., Ghamsari, L., Lasso, G., Honig, B. H., Shapira, S. D., & Wang, H. H. (2016) · PLoS ONE · doi:10.1371/journal.pone.0167617 · Cited by 5
Cited by
Sorted by citations
CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens
Concordet, J., & Haeussler, M. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky354 · Cited by 2,392
Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities
Sanson, K. R., Hanna, R. E., Hegde, M., Donovan, K. F., Strand, C., Sullender, M. E., Vaimberg, E. W., Goodale, A., Root, D. E., Piccioni, F., et al. (2018) · Nature Communications · doi:10.1038/s41467-018-07901-8 · Cited by 1,043
Self-citationZou, Y., Palte, M. J., Deik, A. A., Li, H., Eaton, J. K., Wang, W., Tseng, Y.-Y., Deasy, R., Kost-Alimova, M., Dančík, V., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-09277-9 · Cited by 887
Self-citationCytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis
Zou, Y., Li, H., Graham, E. T., Deik, A., Eaton, J. K., Wang, W., Sandoval-Gomez, G., Clish, C. B., Doench, J. G., & Schreiber, S. L. (2020) · Nature Chemical Biology · doi:10.1038/s41589-020-0472-6 · Cited by 753
Self-citationMimitou, E. P., Cheng, A., Montalbano, A., Hao, S., Stoeckius, M., Legut, M., Roush, T., Herrera, A., Papalexi, E., Ouyang, Z., et al. (2019) · Nature Methods · doi:10.1038/s41592-019-0392-0 · Cited by 520
Metadata
- Authors
- Najm, F. J., Strand, C., Donovan, K. F., Hegde, M., Sanson, K. R., Vaimberg, E. W., Sullender, M. E., Hartenian, E., Kalani, Z., Fusi, N., Listgarten, J., Younger, S. T., Bernstein, B. E., Root, D. E., & Doench, J. G.
- Affiliations
- Broad Institute (US)
- Microsoft Research New England (United States) (US)
- Corresponding author
- Doench, John G
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2017-12-18
- Volume
- 36
- Issue
- 2
- Pages
- 179-189
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC5800952
- OpenAlex
- W2781417302
- Cited by
- 279
- References
- 54
- Citations by year
- 2026: 16 · 2025: 22 · 2024: 26 · 2023: 32 · 2022: 26 · 2021: 45 · 2020: 39 · 2019: 36 · 2018: 37
- FWCI
- 12.51
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- National Cancer Institute (DP1CA216873)
- National Institute of General Medical Sciences (T32GM007226)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- CRISPR/Cas9, SaCas9, SpCas9, single guide RNA design, dual targeting, synthetic lethality, MAPK signaling, apoptosis, TP53, transcriptional activation, lentiviral vectors
- MeSH terms
- Machine Learning, RNA, Guide, CRISPR-Cas Systems, Epistasis, Genetic, Genetic Testing, Humans, Staphylococcus aureus, Streptococcus pyogenes, Signal Transduction, Tumor Suppressor Protein p53, Apoptosis, Gene Targeting, Mitogen-Activated Protein Kinase Kinases, Gene Knockout Techniques, 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
