Paper

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

Curtright, Andrew, Kuang, Xiuli, Schnitzer, Mark, Gradinaru, Viviana, Yuan, Peng, Zeng, Shaoqun, Ouellette, Ben, Kahan, Anat, Cetin, Ali, Gong, Hui, Luo, Qingming, Zeng, Hongkui, Balaram, Pooja, Chrapkiewicz, Radoslaw, Daigle, Tanya L, Tasic, Bosiljka, Yao, Shenqin, Zhou, Thomas, Mortrud, Marty, Chatterjee, Soumya, Wang, Yun, Dhaka, Ajay

2019

Overview

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. Footnotes * With this version of our manuscript we provided proof-of-principle experiments showing that RecV mediated light-inducible site-specific DNA modifications are possible in the mammalian nervous system at single cell level. We provided examples of targeted single-cell 2P-mediated optogenomic modifications and established imaging and conversion parameters to induce such modifications with unprecedented spatial resolution. Our data also provide a quantitative description of the induction specificity of iCreV at different 2P wavelengths and support the feasibility of combining calcium imaging with iCreV system in mice in vivo. We showed that these light-inducible recombinases work highly efficiently and intersectionally in the mouse brain to label specific cell classes or types -SSTs, L5PCs. We further demonstrated that RecVs allow effective light induced optogenomic modifications in multiple loci within the mouse genome and zebrafish. We compared RecV system to other light inducible recombinases in vitro and in vivo. We added new figures and moved some main figures to supplements. We also added new collaborations and authors.

• Publisher: Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
• Subject: Axon guidance / Brain / Calcium imaging / Circuits / Cortex / Cytology / Deoxyribonucleic acid / DNA / Genomes / Light / Nervous system / Neural networks / Neuroimaging / Neuroscience / Recombinase / Recombination / Somatostatin / Spatial discrimination / Structure-function relationships