Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain

Abstract

Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer1,2,3,4,5,6. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics7,8,9,10,11,12,13. Here we describe a capsid selection method, called Cre recombination–based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.

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Figure 1: Cre-dependent recovery of AAV capsid sequences from transduced target cells.
Figure 2: AAV-PHP.B mediates efficient gene delivery throughout the CNS after intravenous injection in adult mice.
Figure 3: AAV-PHP.B transduces multiple CNS cell types more efficiently than AAV9.
Figure 4: AAV-PHP.A exhibits efficient transduction of CNS astrocytes and reduced tropism for peripheral organs.

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References

  1. 1

    Kaplitt, M.G. et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet 369, 2097–2105 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Wu, Z., Asokan, A. & Samulski, R.J. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol. Ther. 14, 316–327 (2006).

    CAS  Article  Google Scholar 

  3. 3

    High, K.H., Nathwani, A., Spencer, T. & Lillicrap, D. Current status of haemophilia gene therapy. Haemophilia 20 (suppl. 4), 43–49 (2014).

    CAS  Article  Google Scholar 

  4. 4

    Borel, F., Kay, M.A. & Mueller, C. Recombinant AAV as a platform for translating the therapeutic potential of RNA interference. Mol. Ther. 22, 692–701 (2014).

    CAS  Article  Google Scholar 

  5. 5

    Ojala, D.S., Amara, D.P. & Schaffer, D.V. Adeno-associated virus vectors and neurological gene therapy. Neuroscientist 21, 84–98 (2015).

    Article  Google Scholar 

  6. 6

    Betley, J.N. & Sternson, S.M. Adeno-associated viral vectors for mapping, monitoring, and manipulating neural circuits. Hum. Gene Ther. 22, 669–677 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Bartlett, J.S., Kleinschmidt, J., Boucher, R.C. & Samulski, R.J. Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab′γ)2 antibody. Nat. Biotechnol. 17, 181–186 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Müller, O.J. et al. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat. Biotechnol. 21, 1040–1046 (2003).

    Article  Google Scholar 

  9. 9

    Grimm, D. et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J. Virol. 82, 5887–5911 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Dalkara, D. et al. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci. Transl. Med. 5, 189ra76 (2013).

    Article  Google Scholar 

  11. 11

    Lisowski, L. et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 506, 382–386 (2014).

    CAS  Article  Google Scholar 

  12. 12

    Maheshri, N., Koerber, J.T., Kaspar, B.K. & Schaffer, D.V. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat. Biotechnol. 24, 198–204 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Excoffon, K.J.D.A. et al. Directed evolution of adeno-associated virus to an infectious respiratory virus. Proc. Natl. Acad. Sci. USA 106, 3865–3870 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Foust, K.D. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 59–65 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Bevan, A.K. et al. Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders. Mol. Ther. 19, 1971–1980 (2011).

    CAS  Article  Google Scholar 

  16. 16

    Maguire, C.A., Ramirez, S.H., Merkel, S.F., Sena-Esteves, M. & Breakefield, X.O. Gene therapy for the nervous system: challenges and new strategies. Neurotherapeutics 11, 817–839 (2014).

    CAS  Article  Google Scholar 

  17. 17

    Gray, S.J. et al. Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol. Ther. 19, 1058–1069 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Maguire, A.M. et al. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N. Engl. J. Med. 358, 2240–2248 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Nathwani, A.C. et al. Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins. Mol. Ther. 19, 876–885 (2011).

    CAS  Article  Google Scholar 

  20. 20

    Gaudet, D. et al. Review of the clinical development of alipogene tiparvovec gene therapy for lipoprotein lipase deficiency. Atheroscler. Suppl. 11, 55–60 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Pulicherla, N. et al. Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer. Mol. Ther. 19, 1070–1078 (2011).

    CAS  Article  Google Scholar 

  22. 22

    Sonntag, F., Schmidt, K. & Kleinschmidt, J.A. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc. Natl. Acad. Sci. USA 107, 10220–10225 (2010).

    CAS  Article  Google Scholar 

  23. 23

    Garcia, A.D.R., Doan, N.B., Imura, T., Bush, T.G. & Sofroniew, M.V. GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat. Neurosci. 7, 1233–1241 (2004).

    CAS  Article  Google Scholar 

  24. 24

    Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).

    CAS  Article  Google Scholar 

  25. 25

    Xie, J. et al. MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol. Ther. 19, 526–535 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Samaranch, L. et al. Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates. Hum. Gene Ther. 23, 382–389 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Dufour, B.D., Smith, C.A., Clark, R.L., Walker, T.R. & McBride, J.L. Intrajugular vein delivery of AAV9-RNAi prevents neuropathological changes and weight loss in Huntington's disease mice. Mol. Ther. 22, 797–810 (2014).

    CAS  Article  Google Scholar 

  28. 28

    Bartlett, J.S., Samulski, R.J. & McCown, T.J. Selective and rapid uptake of adeno-associated virus type 2 in brain. Hum. Gene Ther. 9, 1181–1186 (1998).

    CAS  Article  Google Scholar 

  29. 29

    Wang, H. et al. Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis. Hum. Mol. Genet. 23, 668–681 (2014).

    CAS  Article  Google Scholar 

  30. 30

    Chakrabarty, P. et al. Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain. PLoS One 8, e67680 (2013).

    CAS  Article  Google Scholar 

  31. 31

    Pa¸ca, A.M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).

    Article  Google Scholar 

  32. 32

    Ying, Y. et al. Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library. Gene Ther. 17, 980–990 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Wall, N.R., Wickersham, I.R., Cetin, A., De La Parra, M. & Callaway, E.M. Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus. Proc. Natl. Acad. Sci. USA 107, 21848–21853 (2010).

    CAS  Article  Google Scholar 

  34. 34

    Kawashima, T. et al. Functional labeling of neurons and their projections using the synthetic activity-dependent promoter E-SARE. Nat. Methods 10, 889–895 (2013).

    CAS  Article  Google Scholar 

  35. 35

    Guenthner, C.J., Miyamichi, K., Yang, H.H., Heller, H.C. & Luo, L. Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron 78, 773–784 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Izpisua Belmonte, J.C. et al. Brains, genes, and primates. Neuron 86, 617–631 (2015).

    Article  Google Scholar 

  37. 37

    van der Marel, S. et al. Neutralizing antibodies against adeno-associated viruses in inflammatory bowel disease patients: implications for gene therapy. Inflamm. Bowel Dis. 17, 2436–2442 (2011).

    Article  Google Scholar 

  38. 38

    Calcedo, R., Vandenberghe, L.H., Gao, G., Lin, J. & Wilson, J.M. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J. Infect. Dis. 199, 381–390 (2009).

    Article  Google Scholar 

  39. 39

    Boutin, S. et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum. Gene Ther. 21, 704–712 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Levitt, N., Briggs, D., Gil, A. & Proudfoot, N.J. Definition of an efficient synthetic poly(A) site. Genes Dev. 3, 1019–1025 (1989).

    CAS  Article  Google Scholar 

  41. 41

    Chiorini, J.A., Kim, F., Yang, L. & Kotin, R.M. Cloning and characterization of adeno-associated virus type 5. J. Virol. 73, 1309–1319 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Farris, K.D. & Pintel, D.J. Improved splicing of adeno-associated viral (AAV) capsid protein-supplying pre-mRNAs leads to increased recombinant AAV vector production. Hum. Gene Ther. 19, 1421–1427 (2008).

    CAS  Article  Google Scholar 

  43. 43

    Albert, H., Dale, E.C., Lee, E. & Ow, D.W. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J. 7, 649–659 (1995).

    CAS  Article  Google Scholar 

  44. 44

    Shaner, N.C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

    CAS  Article  Google Scholar 

  45. 45

    Hancock, J.F., Cadwallader, K., Paterson, H. & Marshall, C.J. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 10, 4033–4039 (1991).

    CAS  Article  Google Scholar 

  46. 46

    Gray, S.J. et al. Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration. Curr. Protoc. Neurosci. S57, 4.17.1–4.17.30 (2011).

    Article  Google Scholar 

  47. 47

    Ayuso, E. et al. High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther. 17, 503–510 (2010).

    CAS  Article  Google Scholar 

  48. 48

    Zolotukhin, S. et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973–985 (1999).

    CAS  Article  Google Scholar 

  49. 49

    Wobus, C.E. et al. Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection. J. Virol. 74, 9281–9293 (2000).

    CAS  Article  Google Scholar 

  50. 50

    Treweek, J.B. et al. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat. Protoc. 10, 1860–1896 (2015).

    CAS  Article  Google Scholar 

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Acknowledgements

This article and the naming of the novel AAV clones are dedicated to the memory of Paul H. Patterson (P.H.P.), who passed away during the preparation of this manuscript. We wish to thank L. Rodriguez and P. Anguiano for administrative assistance, A. Balazs and S. Cassenaer and the entire Gradinaru and Patterson laboratories for helpful discussions, and A. Choe for helpful comments on the manuscript. We thank the University of Pennsylvania vector core for the AAV2/9 Rep-Cap plasmid, A. Balazs and D. Baltimore for the AAV genome plasmid, and the Biological Imaging Facility, supported by the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation, for use of imaging equipment. This work was supported by grants from the Hereditary Disease Foundation and the Caltech–City of Hope Biomedical Initiative (to P.H.P.) and from the National Institutes of Health (NIH) Director's New Innovator 1DP2NS087949; NIH/National Institute on Aging (NIA) 1R01AG047664; Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research; and the Gordon and Betty Moore Foundation through grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative (to V.G.). Work in the Gradinaru laboratory is also funded by the following awards (to V.G.): NIH BRAIN 1U01NS090577; NIH/National Institute of Mental Health (NIMH) 1R21MH103824-01; Pew Charitable Trust; Sloan Foundation; Kimmel Foundation; Human Frontiers in Science Program; Caltech-GIST; Caltech–City of Hope Biomedical Initiative. Work in the Pasca laboratory is supported by a NIMH 1R01MH100900 and 1R01MH100900-02S1, the NIMH BRAINS Award (R01MH107800), the California Institute of Regenerative Medicine (CIRM), the MQ Fellow Award and the Donald E. and Delia B. Baxter Foundation Scholar Award (to S.P.P.).

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B.E.D. designed and performed experiments, analyzed data, prepared figures and wrote the manuscript. P.L.P., B.P.S., S.R.K., A.B. and K.Y.C. performed experiments, virus production and characterization. W.-L.W. performed tissue processing and IHC. B.Y. assisted with tissue clearing and imaging. N.H. and S.P.P. performed the experiments with human cells, analyzed the data, and prepared the associated figure and text. V.G. helped with study design and data analysis, manuscript and figure preparation and supervised the project. All authors edited and approved the manuscript.

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Correspondence to Benjamin E Deverman or Viviana Gradinaru.

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The California Institute of Technology has filed patent applications related to this work with B.E.D, B.Y. and V.G. listed as inventors.

Integrated supplementary information

Supplementary Figure 1 Capsid library fragment generation and Cre-dependent capsid sequence recovery.

(a) Schematic shows PCR products (yellow bar) with 7AA of randomized sequence (represented by the full spectrum vertical bar) inserted after amino acid 588. The primers used to generate the library are indicated by name and half arrows. The PCR template was modified to eliminate a naturally occurring EarI restriction site within the capsid gene fragment (xE) (See Online Methods for details). (b) The schematic shows the rAAV-Cap-in-cis genome and the primers used to quantify vector genomes (left) and recover sequences that have transduced Cre expressing cells (right). The PCR-based recovery is performed in two steps. Step 1 (blue) provides target cell-specific sequence recovery by selectively amplifying Cap sequences from genomes that have undergone Cre-dependent inversion of the downstream polyadenylation (pA) sequence. For step 1, 9CapF functions as a forward primer and the CDF primer functions as the reverse primer on templates recombined by Cre. Step 2 (magenta) uses primers XF and AR to generate the PCR product that is cloned into rAAV-ΔCap-in-cis plasmid (library regeneration) or to clone into an AAV2/9 rep-cap trans plasmid (variant characterization). (c) The table provides the sequences for the primers shown in a and b.

Supplementary Figure 2 The most enriched variants recovered from in vivo selections in GFAP-Cre mice.

(a) The table provides the 7-mer AA and nucleic acid sequences, percentage enrichment (% of total variants sequenced), capsid characteristics, and production efficiencies of the three most enriched variants from each selection. (b) Images of representative sagittal brain sections from mice assessed 2 weeks after injection of 3.3x1010 vg/mouse of ssAAV-CAG-mNeonGreen-farnesylated (mNeGreen-f) packaged into AAV-PHP.B or the second or third most enriched variants, AAV-PHP.B2 and AAV-PHP.B3. Data are representative of 2 (AAV-PHP.B) and 3 (AAV-PHP.B2 and AAV-PHP.B3) mice per group. (c) DNase-resistant vg obtained from preparations of the individual variants recovered from GFAP-Cre selections. Yields are given as the number of purified vector genome (vg) copies per 150 mm dish of producer cells; mean ± s.d. *p<0.05, one-way ANOVA and Tukey’s multiple comparison test. The number of independent preparations for each capsid is shown within the bar.

Supplementary Figure 3 AAV-PHP.B transduces several interneuron cell types and endothelial cells but does not appear to transduce microglia.

(a-d) Adult mice were injected with 1x1012 vg of AAV-PHP.B:CAG-GFP and assessed for GFP expression 3 weeks later. Representative images show IHC for GFP (a-c) or native GFP fluorescence (d) in green together with IHC for the indicated antigen (magenta) and brain region. (e) Adult mice were injected with 3.3x1010 vg of ssAAV-PHP.B:CAG-mNeGrn-f and assessed at 2 weeks post injection. Native fluorescence from mNeGrn-f co-localizes with some endothelial cells expressing CD31. (f, g) Adult mice were injected with 2x1012 vg of ssAAV-PHP.B:CAG-NLS-GFP and assessed at 3 weeks post injection. Images show native NLS-GFP expression along with Iba1. Asterisks indicate cells that express the indicated antigen, but not detectable GFP. Parvalbumin (PV), Calbindin (Calb) and Calretinin (CR). Scale bars: 20 μm (a-d); 50 μm (e, f) and 500 μm (g).

Supplementary Figure 4 Long-term eGFP expression in the brain following gene transfer with AAV-PHP.B.

Adult mice were intravenously injected with the indicated dose of ssAAV-CAG-GFP packaged into AAV9 or AAV-PHP.B and were assessed for native eGFP fluorescence 377 Days later. N=1 per vector/dose.

Supplementary Figure 5 Representative images of native GFP fluorescence and IHC for several neuron and glial cell types following transduction by AAV-PHP.B:CAG-NLS-GFP.

(a-d) Adult mice were injected with 2x1012 vg of ssAAV-PHP.B:CAG-NLS-GFP and assessed at 3 weeks post injection. Images show native NLS-GFP expression along with IHC for the indicated antigen in the indicated brain region. In all panels, arrows indicate co-localization of GFP expression with IHC for the indicated antigen. (b, c) Single-plane confocal images; (a, d) MIP. Corpus callosum (cc), substantia nigra pars compacta (SNc), ventral tegmental area (VTA). Scale bars: 50 μm.

Supplementary Figure 6 AAV9, AAV-PHP.A and AAV-PHP.B transduce human iPSC-derived cortical neurons and astrocytes in dissociated cultures and intact 3D cortical cultures.

(a) AAV-PHP.B provides higher transduction of human neurons and astrocytes in dissociated monolayer cultures. Representative images show GFP expression at five days after viral transduction (ssAAV-CAG-NLS-GFP packaged in AAV9, AAV-PHP.A or AAV-PHP.B; 1x109 vg/well) of dissociated iPSC-derived human cortical spheroids differentiated in vitro. GFP expressing cells (green) co-localize with astrocytes immunostained for GFAP (cyan) or neurons immunostained for MAP2 (magenta) as indicated by white arrows. (b) Quantification of the percentage of GFAP+ or MAP2+ cells transduced by AAV9, AAV-PHP.A or AAV-PHP.B (n=3 differentiations into cortical spheroids of two human iPSC lines derived from two subjects; two-way ANOVA, Tukey’s multiple comparison test; mean ± s.d). (c) AAV9, AAV-PHP.A and AAV-PHP.B transduce intact human 3D cortical cultures (cortical spheroids differentiated from human iPSCs). Images of human iPSC-derived cortical spheroid cryosections (day 205 of in vitro differentiation) transduced with ssAAV-CAG-NLS-GFP packaged in AAV9, AAV-PHP.A or AAV-PHP.B show native GFP fluorescence together with immunostaining for GFAP and MAP2. Insets show co-labeling of GFP fluorescence with GFAP+ astrocytes (cyan) or MAP2+ (magenta) neurons. Scale bars: 40 μm (a); 100 μm (c).

Supplementary Figure 7 AAV-PHP.B and AAV-PHP.A capsids localize to the brain vasculature after intravenous injection and transduce cells along the vasculature by 24 hours post-administration.

Adult mice were injected with 1x1011 vg of ssAAV-CAG-NLS-GFP packaged into AAV9, AAV-PHP.A or AAV-PHP.B as indicated. (a, b) Representative images of capsid immunostaining (green) using the B1 anti-AAV VP3 antibody that recognizes a shared internal epitope in the cerebellum (a) or striatum (b) in the brains of mice injected intravenously one hour prior to fixation by cardiac perfusion. Cell nuclei are labeled with DAPI (magenta). Lipofuscin autofluorescence (yellow) can be distinguished from capsid staining by its presence in both green and red channels. The inset (right) shows a 3D MIP image of the area highlighted in the AAV-PHP.B image. Arrows highlight capsid IHC signal; asterisks indicate vascular lumens. Data are representative of 2 (no virus and AAV-PHP.A) or 3 (AAV9 and AAV-PHP.B) mice per group. (c) Representative image of GFP expression (green) with DAPI (white) and CD31 (magenta) 24 hours post-administration of AAV-PHP.B. Arrows highlight GFP-expressing cells. (d) Quantification of the number of GFP expressing cells present along the vasculature in the indicated brain regions. N=3 per group; mean ± s.d; AAV-PHP.B vs AAV9 and AAV-PHP.A, ***p<0.001 for all regions; AAV9 vs AAV-PHP.A, not significant; two-way ANOVA. Scale bars: 200 μm (a); 50 μm (b, c); Major tick marks are 50 μm in the high magnification inset (a).

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Deverman, B., Pravdo, P., Simpson, B. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204–209 (2016). https://doi.org/10.1038/nbt.3440

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