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

Yao, Shenqin and Yuan, Peng and Ouellette, Ben and Zhou, Thomas and Mortrud, Marty and Balaram, Pooja and Chatterjee, Soumya and Wang, Yun and Daigle, Tanya L. and Tasic, Bosiljka and Kuang, Xiuli and Gong, Hui and Luo, Qingming and Zeng, Shaoqun and Curtright, Andrew and Dhaka, Ajay and Kahan, Anat and Gradinaru, Viviana and Chrapkiewicz, Radosław and Schnitzer, Mark and Zeng, Hongkui and Cetin, Ali (2019) RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations. . (Unpublished)

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Brain circuits are composed of vast numbers of intricately interconnected neurons with diverse molecular, anatomical and physiological properties. To allow highly specific “user-defined” targeting of individual neurons for structural and functional studies, we modified three site-specific DNA recombinases, Cre, Dre and Flp, by combining them with a fungal light-inducible protein, Vivid, to create light-inducible recombinases (named RecV). We generated viral vectors to express these light-inducible recombinases and demonstrated that they can induce genomic modifications in dense or sparse populations of neurons in superficial as well as deep brain areas of live mouse brains by one-photon or two-photon light induction. These light-inducible recombinases can produce highly targeted, sparse and strong labeling of individual neurons in multiple loci and species. They can be used in combination with other genetic strategies to achieve specific intersectional targeting of mouse cortical layer 5 or inhibitory somatostatin neurons. In mouse cortex sparse light-induced recombination allows whole-brain morphological reconstructions to identify axonal projection specificity. Furthermore these enzymes allow single cell targeted genetic modifications via soma restricted two-photon light stimulation in individual cortical neurons and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally-precise, targeted optogenomic modifications that could greatly facilitate detailed analysis of neural circuits at the single cell level by linking genetic identity, morphology, connectivity and function.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper
Gradinaru, Viviana0000-0001-5868-348X
Alternate Title:RecV recombinase system for spatiotemporally controlled light-inducible genomic modifications
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. We are grateful to the Structured Science teams at the Allen Institute for their technical support in stereotaxic injections and mouse colony management. The work was funded by the Allen Institute for Brain Science, NIMH BRAIN Initiative grant RF1MH114106 to A.C., NSFC Science Fund for Creative Research Group of China (Grant No. 61421064) to H.G., Q.L. and S.Z., NIH Director's New Innovator award IDP20D017782 and NIH/NIA 1R01AG047664 to V.G., and Colvin divisional fellowship of Division of Biology and Biological Engineering, California Institute of Technology, to A.K. NIH Brain Initiative U01NS107610 grant to Mark Schnitzer. Immunohistochemistry experiments in Figure 8 were performed in the Biological Imaging Facility, with the support of the California Institute of Technology Beckman Institute and the Arnold and Mabel Beckman Foundation. The creation of Ai139 mouse line was supported by the NIH grant R01DA036909 to B.T. The authors thank Sevi Durdu, Bilal Kerman and Keisuke Yonehara for critical reading and feedback. The authors wish to thank the Allen Institute founder, Paul G. Allen, for his vision, encouragement, and support. AUTHOR CONTRIBUTIONS: A.C. conceptualized the light-inducible recombinase system. S.Y. performed cloning and characterization of the constructs as well as participated in image acquisition. B.O. performed all the surgeries and image acquisition. T.Z. Performed cloning. M.M. performed some of the surgeries and light stimulations. T.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. performed deep brain imaging experiments. S.C. and P.B. performed 2P induced recombination experiments. A.CT. and A.D. performed zebrafish experiments. R.C., P.Y and M.S. performed the targeted single cell 2P experiments and combinatorial cortical GCamp7F calcium imaging experiments. A.C. and H.Z. designed and coordinated the study as well as wrote the manuscript, with inputs from all coauthors. Data availability: All relevant plasmids will be deposited to Addgene. Data are available from the corresponding author upon request. The authors declare no competing financial interests.
Funding AgencyGrant Number
Allen Institute for Brain ScienceUNSPECIFIED
National Natural Science Foundation of China61421064
Caltech Division of Biology and Biological EngineeringUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
Subject Keywords:Light-inducible, optogenomic, recombinase, recombination, RecV, Vivid, Cre, Dre, Flp
Record Number:CaltechAUTHORS:20190220-102621411
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Official Citation:RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations Shenqin Yao, Peng Yuan, Ben Ouellette, Thomas Zhou, Marty Mortrud, Pooja Balaram, Soumya Chatterjee, Yun Wang, Tanya L. Daigle, Bosiljka Tasic, Xiuli Kuang, Hui Gong, Qingming Luo, Shaoqun Zeng, Andrew Curtright, Ajay Dhaka, Anat Kahan, Viviana Gradinaru, Radosław Chrapkiewicz, Mark Schnitzer, Hongkui Zeng, Ali Cetin bioRxiv 553271; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:97389
Deposited By: George Porter
Deposited On:24 Jul 2019 21:58
Last Modified:24 Jul 2019 21:58

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