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Targeting cells with single vectors using multiple-feature Boolean logic

Nature Methods volume 11, pages 763–772 (2014)Cite this article

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Abstract

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.

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Figure 1: Diversifying recombinase targeting strategies.
Figure 2: Intron engineering for INTRSECT.
Figure 3: INTRSECT: recombinases with engineered introns enable intersectional targeting in vitro.
Figure 4: INTRSECT specificity and functionality in vivo.
Figure 5: Exclusion logic using INTRSECT.
Figure 6: Combinatorial targeting with multiplexed recombinases.

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Acknowledgements

We thank M. Kay (Stanford) for providing the pRC-DJ plasmid used to produce AAV-DJ; M. Bigos at the Stanford Shared FACS Facility for assistance with experiment planning and data analysis; S. Roy for extensive discussions regarding intron function and structure; P. Wu for technical assistance in mouse engineering and characterization; and the entire Deisseroth lab for helpful discussions. The Neuroscience Gene Vector and Virus Core at Stanford is funded in part by US National Institutes of Health grant # NINDS P30 NS069375-01A1. This work is supported by the Stanford Medical Scientist Training Program (MSTP; L.E.F. and J.M.), a Stanford Bio-X Fellowship (J.M. and M. Hyun), the Samsung Scholarship (M. Hyun), the Robertson Neuroscience Fund of Cold Spring Harbor Laboratory (M. He, J.T. and Z.J.H.), the Stony Brook University MSTP (J.T.), a Howard Hughes Medical Institute International Student Fellowship (A.S.), National Science Foundation (NSF) grant 0801700 (L.G.), the NSF Graduate Research Fellowship Program (K.A.Z.), the New York University MSTP (H.B.), the Human Frontiers Science Project and German Academic Exchange Service (DAAD; I.D.), and US National Institute on Drug Abuse (NIDA) DA024763 (C.E.B.). K.D. is supported by the Wiegers Family Fund, US National Institute of Mental Health, NIDA, Defense Advanced Research Projects Agency REPAIR Program, Keck Foundation, McKnight Foundation, Gatsby Charitable Foundation, Snyder Foundation, Woo Foundation, Tarlton Foundation, and Albert Yu and Mary Bechman Foundation. All tools and methods are distributed and supported freely at http://www.optogenetics.org/ and Addgene.

Author information

Author notes

  1. Lief E Fenno, Joanna Mattis and Charu Ramakrishnan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroscience, Stanford University, Stanford, California, USA

    Lief E Fenno, Joanna Mattis, Aslihan Selimbeyoglu, Logan Grosenick & Kelly A Zalocusky

  2. Department of Bioengineering, Stanford University, Stanford, California, USA

    Lief E Fenno, Joanna Mattis, Charu Ramakrishnan, Minsuk Hyun, Soo Yeun Lee, Andre Berndt, Logan Grosenick, Ilka Diester & Karl Deisseroth

  3. CNC Program, Stanford University, Stanford, California, USA

    Soo Yeun Lee, Logan Grosenick, Kelly A Zalocusky, Haley Swanson, Chelsey Perry & Karl Deisseroth

  4. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Miao He, Jason Tucciarone & Z Josh Huang

  5. Program in Neuroscience, Stony Brook University, Stony Brook, New York, USA

    Jason Tucciarone

  6. Department of Neuroscience and Physiology, New York University School of Medicine, New York, New York, USA

    Hannah Bernstein

  7. Ernst Struengmann Institute for Neuroscience, Frankfurt, Germany

    Ilka Diester

  8. Department of Neurology, Massachusetts General Hospital, Cambridge, Massachusetts, USA

    Frederick M Boyce

  9. Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, USA

    Caroline E Bass

  10. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Rachael Neve

  11. Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA

    Karl Deisseroth

  12. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Karl Deisseroth

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  1. Lief E Fenno
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Contributions

L.E.F., J.M., C.R. and K.D. designed the study and interpreted results. L.E.F., J.M. and K.D. wrote the paper. L.E.F. and J.M. coordinated the experiments. L.E.F., J.M., S.Y.L., A.B. and A.S. performed in vitro electrophysiology experiments. J.M. and K.A.Z. performed in vivo electrophysiology experiments. L.E.F., J.M., M. Hyun, A.S., K.A.Z., H.B. and I.D. performed viral injections. L.E.F., J.M., M. Hyun, S.Y.L., J.T., K.A.Z., H.B., H.S., I.D. and C.P. performed immunohistochemistry. L.E.F., C.R., R.N., C.E.B. and F.M.B. provided viruses. L.G., J.M. and L.E.F. performed statistical analysis. L.E.F., J.M., C.R., M. Hyun, A.B. and C.P. performed molecular engineering and characterization. M. He, J.T. and Z.J.H. provided animals. All authors contributed to editing. K.D. supervised all aspects of the project.

Corresponding author

Correspondence to Karl Deisseroth.

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Fenno, L., Mattis, J., Ramakrishnan, C. et al. Targeting cells with single vectors using multiple-feature Boolean logic. Nat Methods 11, 763–772 (2014). https://doi.org/10.1038/nmeth.2996

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