Cas9 is an endonuclease that can be programed to autonomously deliver diverse effectors to specified genetic addresses. High-resolution structures of this protein and its associated CRISPR RNA guide explain the molecular mechanisms of CRISPR-RNA-guided DNA recognition and provide a molecular blueprint that has facilitated structure-guided functional remodeling. Here we retrace events that led from early efforts to understand the central role of Cas9 in CRISPR-mediated adaptive immunity to contemporary efforts aimed at developing and deploying this enzyme for programmable genetic editing.
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Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).
Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005).
Mojica, F. J., Díez-Villaseñor, C., García-Martínez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008).
Horvath, P. et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190, 1401–1412 (2008).
Mojica, F. J., Díez-Villaseñor, C., García-Martínez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).
Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).
Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). Cas9 is a dual-RNA-guided endonuclease that creates dsDNA-breaks, and the two RNAs can be linked by a tetraloop into a chimeric single-guide RNA.
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014). This paper provides the first crystal structure of SpCas9 without guide RNA or target DNA.
Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016). This crystal structure of active Cas9 with sgRNA and 30-bp double-strand target DNA reveals the rotation of the NHN domain toward the DNA.
Jiang, F., Zhou, K., Ma, L., Gressel, S. & Doudna, J. A. A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015). The crystal structure of sgRNA-bound active Cas9 shows extensive protein rearrangement upon RNA binding and preordering of the 10-nt seed region.
Nishimasu, H. et al. Crystal structure of Staphylococcus aureus Cas9. Cell 162, 1113–1126 (2015).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014). This study provides the crystal structure of Cas9 in complex with an sgRNA and a 23-nt single-strand target DNA.
Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014). This study provides the crystal structure of sgRNA-Cas9 bound to a complementary DNA strand and a duplexed PAM. This structure explains the mechanism of PAM recognition.
Hirano, H. et al. Structure and engineering of Francisella novicida Cas9. Cell 164, 950–961 (2016).
Yamada, M. et al. Crystal structure of the minimal Cas9 from Campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 systems. Mol. Cell 65, 1109–1121.e3 (2017).
Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014).
Shibata, M. et al. Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy. Nat. Commun. 8, 1430 (2017).
Osuka, S. et al. Real-time observation of flexible domain movements in CRISPR-Cas9. EMBO J. 37, e96941 (2018).
Palermo, G., Miao, Y., Walker, R. C., Jinek, M. & McCammon, J. A. CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations. Proc. Natl. Acad. Sci. USA 114, 7260–7265 (2017).
Sundaresan, R., Parameshwaran, H. P., Yogesha, S. D., Keilbarth, M. W. & Rajan, R. RNA-independent DNA cleavage activities of Cas9 and Cas12a. Cell Reports 21, 3728–3739 (2017).
Wright, A. V. et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl. Acad. Sci. USA 112, 2984–2989 (2015).
Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014). This paper clarifies the mechanism target identification, showing that the search process is primarily governed by three-dimensional diffusion.
Globyte, V. et al. CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion. EMBO J. (in the press).
Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 527, 110–113 (2015). These authors demonstrate that complete base pairing to the target is an allosteric regulator of the HNH and RuvC nuclease domains.
Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A. & Ha, T. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat. Commun. 7, 12778 (2016).
Yang, M. et al. The conformational dynamics of Cas9 governing DNA cleavage are revealed by single-molecule FRET. Cell Reports 22, 372–382 (2018).
Singh, D. et al. Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis. Nat. Struct. Mol. Biol. 25, 347–354 (2018).
Gong, S., Yu, H. H., Johnson, K. A. & Taylor, D. W. DNA unwinding is the primary determinant of CRISPR-Cas9 activity. Cell Reports 22, 359–371 (2018).
Yourik, P., Fuchs, R.T., Mabuchi, M., Curcuru, J.L. & Robb, G.B. Staphylococcus aureus Cas9 is a multiple-turnover enzyme. RNA rna.067355.118 (2018).
Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Hirano, S., Nishimasu, H., Ishitani, R. & Nureki, O. Structural basis for the altered PAM specificities of engineered CRISPR-Cas9. Mol. Cell 61, 886–894 (2016).
Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).
Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).
Lin, L. et al. Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells. J. Biotechnol. 247, 42–49 (2017).
Shao, S. et al. Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52. Int. J. Biochem. Cell Biol. 92, 43–52 (2017).
Charpentier, M. et al. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat. Commun. 9, 1133 (2018).
Gutschner, T., Haemmerle, M., Genovese, G., Draetta, G. F. & Chin, L. Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Reports 14, 1555–1566 (2016).
Lee, K. et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. eLife 6, e25312 (2017).
Carlson-Stevermer, J. et al. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat. Commun. 8, 1711 (2017).
Savic, N. et al. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. eLife 7, e33761 (2018).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). Cas9 fusion to rat APOBEC1 cytidine deaminase and the uracil glycosylase inhibitor enables programmable base editing in cells without dsDNA cleavage.
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016). Fusion of Cas9 to activation-induced cytidine deaminase and the uracil glycosylase inhibitor enables programmable base editing in cells without dsDNA cleavage.
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).
Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).
Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437 (2013).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013). There are two main points in this paper: insertion of aptamer sequences into the guide-RNA enables site-specific recruitment of transcriptional activators; and paired Cas9 nickases reduce off-target cleavage activity.
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013). This study provides the first demonstration of using nuclease inactive Cas9 (dCas9) for transcriptional repression in bacteria.
Dahlman, J. E. et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 33, 1159–1161 (2015).
Kiani, S. et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015).
Paradise, B. D., Barham, W. & Fernandez-Zapico, M. E. Targeting epigenetic aberrations in pancreatic cancer, a new path to improve patient outcomes?. Cancers (Basel) 10, E128 (2018).
Song, S. & Johnson, F. B. Epigenetic mechanisms impacting aging: a focus on histone levels and telomeres. Genes (Basel) 9, E201 (2018).
Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).
Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).
Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat. Methods 12, 401–403 (2015). This study provides the first demonstration of dCas9-targeted epigenetic modification using the LSD1 histone demethylase to define enhancer elements in mouse embryonic stem cells.
Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015). This paper demonstrated that dCas9 can be used control cellular phenotypes by targeted manipulation of histone acetylation.
Kwon, D. Y., Zhao, Y. T., Lamonica, J. M. & Zhou, Z. Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC. Nat. Commun. 8, 15315 (2017).
Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018).
Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018).
Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32, 677–683 (2014).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs. Genome Res. 28, 1193–1206 (2018).
Xu, X. & Qi, L. S. A CRISPR-dCas toolbox for genetic engineering and synthetic biology. J. Mol. Biol. S0022-2836(18), 30666–1 (2018).
Lino, C. A., Harper, J. C., Carney, J. P. & Timlin, J. A. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 25, 1234–1257 (2018).
Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).
Fine, E. J. et al. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci. Rep. 5, 10777 (2015).
Truong, D. J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).
Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A. & Liu, D. R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).
Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H. & Sato, M. CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).
Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).
Balboa, D. et al. Conditionally stabilized dCas9 activator for controlling gene expression in human cell reprogramming and differentiation. Stem Cell Reports 5, 448–459 (2015).
Maji, B. et al. Multidimensional chemical control of CRISPR-Cas9. Nat. Chem. Biol. 13, 9–11 (2017).
Liu, K. I. et al. A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing. Nat. Chem. Biol. 12, 980–987 (2016).
Müller, K. et al. A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells. Nucleic Acids Res. 41, e77 (2013).
Shao, J. et al. Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation. Proc. Natl. Acad. Sci. USA 115, E6722–E6730 (2018).
Bubeck, F. et al. Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. Nat. Methods 15, 924–927 (2018).
Shin, J. et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci. Adv. 3, e1701620 (2017).
Yang, H. & Patel, D. J. Inhibition mechanism of an anti-CRISPR suppressor AcrIIA4 targeting SpyCas9. Mol. Cell 67, 117–127.e5 (2017).
Rouet, R. et al. Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing. J. Am. Chem. Soc. 140, 6596–6603 (2018).
Davies, J. O., Oudelaar, A. M., Higgs, D. R. & Hughes, J. R. How best to identify chromosomal interactions: a comparison of approaches. Nat. Methods 14, 125–134 (2017).
Wang, S., Su, J. H., Zhang, F. & Zhuang, X. An RNA-aptamer-based two-color CRISPR labeling system. Sci. Rep. 6, 26857 (2016).
Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
Morgan, S. L. et al. Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nat. Commun. 8, 15993 (2017).
Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).
Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).
Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).
Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).
Chen, J. S. et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550, 407–410 (2017).
Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Lim, Y. et al. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease. Nat. Commun. 7, 13350 (2016).
Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A. & Yildiz, A. A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9. Sci. Adv. 3, o0027 (2017).
Rueda, F. O. et al. Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease. Nat. Commun. 8, 1610 (2017).
Yin, H. et al. Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. Nat. Chem. Biol. 14, 311–316 (2018).
Rahdar, M. et al. Synthetic CRISPR RNA-Cas9-guided genome editing in human cells. Proc. Natl. Acad. Sci. USA 112, E7110–E7117 (2015).
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).
Cromwell, C. R. et al. Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity. Nat. Commun. 9, 1448 (2018).
Clarke, R. et al. Enhanced bacterial immunity and mammalian genome editing via RNA-polymerase-mediated dislodging of Cas9 from double-strand DNA breaks. Mol. Cell 71, 42–55.e8 (2018).
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).
Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol.36, 977–982 (2018).
Heler, R. et al. Mutations in Cas9 enhance the rate of acquisition of viral spacer sequences during the CRISPR-Cas immune response. Mol. Cell 65, 168–175 (2017).
Heler, R. et al. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature 519, 199–202 (2015).
Wei, Y., Terns, R. M. & Terns, M. P. Cas9 function and host genome sampling in Type II-A CRISPR-Cas adaptation. Genes Dev. 29, 356–361 (2015).
Borges, A. L., Davidson, A. R. & Bondy-Denomy, J. The discovery, mechanisms, and evolutionary impact of anti-CRISPRs. Annu. Rev. Virol. 4, 37–59 (2017).
Pawluk, A., Davidson, A. R. & Maxwell, K. L. Anti-CRISPR: discovery, mechanism and function. Nat. Rev. Microbiol. 16, 12–17 (2018).
Carter, J., Hoffman, C. & Wiedenheft, B. The interfaces of genetic conflict are hot spots for innovation. Cell 168, 9–11 (2017).
Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017). This paper provides an evolutionary road map for CRISPR-Cas classification.
Bondy-Denomy, J. et al. A unified resource for tracking anti-CRISPR names. The CRISPR Journal 1, 304–305 (2018).
Pawluk, A. et al. Naturally occurring off-switches for CRISPR-Cas9. Cell 167, 1829–1838.e9 (2016).
Rauch, B. J. et al. Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell 168, 150–158.e10 (2017).
Hynes, A. P. et al. Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nat. Commun. 9, 2919 (2018).
Research in the Wiedenheft lab is supported by the National Institutes of Health (P20GM103500, P30GM110732, R01GM110270, R01GM108888, and R21 AI130670), the National Science Foundation EPSCoR (EPS-110134), the M. J. Murdock Charitable Trust, a young investigator award from Amgen, and the Montana State University Agricultural Experimental Station (USDA NIFA).
B.W. is the founder of SurGene LLC and is an inventor on patent applications related to CRISPR-Cas systems and applications thereof.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ligand-induced conformational changes of Cas9. The movie shows a series of conformational changes starting with a structure of Cas9 protein from Streptococcus pyogenes (SpCas9, PDB ID 4CMP) that transitions to the sgRNA-bound conformation (4ZT0), the ssDNA bound state (4008), PAM-bound state (4UN3), and finally to a complex that includes dsDNA + PAM (5F9R). Positions of the HNH and RuvC nuclease domains and their active sites are highlighted. The movie was created using ChimeraX (Protein Sci. 27, 14–25, 2018).
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Wilkinson, R.A., Martin, C., Nemudryi, A.A. et al. CRISPR RNA-guided autonomous delivery of Cas9. Nat Struct Mol Biol 26, 14–24 (2019). https://doi.org/10.1038/s41594-018-0173-y
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