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Published April 2020 | Supplemental Material + Submitted
Journal Article Open

RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations


Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.

Additional Information

© 2020 Springer Nature Limited. Received 13 January 2018; Accepted 11 February 2020; Published 23 March 2020. We are grateful to the Structured Science teams at the Allen Institute for technical support with stereotaxic injections and mouse colony management. The work was funded by the Allen Institute for Brain Science; NIMH BRAIN Initiative grant no. RF1MH114106 to A.Cetin; the NSFC Science Fund for Creative Research Group of China (grant 61721092) to H.G., Q.L. and S.Z.; NIH Brain Initiative grant no. RF1MH117069 to V.G.; the Colvin divisional fellowship of the Division of Biology and Biological Engineering, California Institute of Technology, to A.K.; and NIH BRAIN Initiative grant U01NS107610 to M.S. The creation of the Ai139 mouse line was supported by NIH grant no. R01DA036909 to B.T. We thank S. Durdu, H. Bayer, D. Schrom, B. Kerman and K. Yonehara for critical reading and feedback. We thank the Allen Institute founder, P.G. Allen, for his vision, encouragement and support. Data availability: DNA sequences of the NCreV, CCreV, NDreV, CDreV, iCreV, iDreV and iFlpV created in this work are curated in National Institute of Health, GenBank. Accession codes are NCreV, MT036266; CCreV, MT036267; NDreV, MT036268; CDreV, MT036269; iCreV, MN944913; iFlpV, MN944914; and iDreV, MN944915. AAV iCreV, 140135; AAV iDreV, 140136; AAV iFlpV, 140137; AAV NCreV, 140131; AAV CCreV, 140132; AAV NDreV, 140134; and AAV CDreV, 140133. Plasmids have been deposited in Addgene with the indicated accession codes. All in vivo and in vitro raw data images used in all figures presented in the paper are available from the corresponding author upon request. Source data files for all figures with graphs are provided in raw tabular form as Excel files. Code availability: The two-photon microscope was operated using ScanImage v.5.3 (Vidrio Technologies, LLC) software and custom software written in LabView 2015 (National Instruments). The code is available upon request to the authors. Author Contributions: A. Cetin conceptualized the light-inducible recombinase system. S.Y. performed cloning and characterization of the constructs and participated in image acquisition. B.O. and P.B. performed surgeries, immunohistochemistry and image acquisition. T.Z. Performed cloning. M.M. performed some of the surgeries and light stimulations. T.L.D. performed some of the initial cloning experiments. B.T. and H.Z. contributed to the generation of the Ai139 transgenic mice. H.G., Q.L. and S.Z. acquired fMOST data. X.K. and Y.W. performed Neurolucida reconstructions. V.G. and A.K. designed deep brain imaging experiments and generated the associated data, figure and text. A.K. performed deep brain imaging experiments. S.C. and P.B. performed 2P-induced recombination experiments. A. Curtright and A.D. performed zebrafish experiments. R.C., P.Y. and M.S. performed targeted single-cell 2P experiments and combinatorial cortical jGCaMP7F calcium imaging experiments. A. Cetin and H.Z. designed and coordinated the study, and wrote the manuscript with inputs from all co-authors. The authors declare no competing interests.

Attached Files

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Supplemental Material - 41592_2020_774_Fig10_ESM.webp

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August 22, 2023
December 22, 2023