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

Abstract

Brain circuits comprise vast numbers of intricately interconnected neurons with diverse molecular, anatomical and physiological properties. To allow “user-defined” targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases (SSRs) 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.

Controlling gene expression with light: a multidisciplinary endeavour

“…Chemical modification of nucleic acids with photocages has been used to create conductive pathways in synthetic tissues [ 37 , 38 ] and control the development of zebrafish [ 7 , 55 , 60 , 134 , 135 ] and cancer therapeutics [ 75 , 76 , 79 , 137 ] with light. While naturally light-sensitive proteins have been used to control cell division [ 99 ], bioreactors [ 129 ], and genome activation in zebrafish and mice [ 146 , 151 , 153 ]. Identification of new applications is vital to realising the full potential of light-controlled gene expression.…”

“…However, in bacteria and yeast these systems can be controlled over multiple generations as the plasmid system encoding the light-sensitive proteins is replicated. An important goal for this field is to combine the light-activation systems with cell delivery systems, where only limited examples exist [ 53 , 72 , 73 , 78 , 146 , 151 , 153 ], to produce far-reaching technologies.…”

Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution

“…generated light-inducible VVD-based Cre, Dre, and Flp recombinase system denoted as RecV. After inducing by one-photon or two-photon light illumination, RecV enables spatiotemporally precise optogenomic modifications of single cells or cell populations in mouse and zebrafish ( 97 ).…”

Neurobiology of cannabinoid receptor signaling

“…In further developments, high-resolution and specific genetic manipulations can be included, such as intersectional targeting of cells and circuits, [178][179][180] and optogenetic-induced genetic manipulation. 181 Furthermore, with the advent of the clustered regularly interspaced short palindromic sequences (CRISPR)/ CRISPR-associated protein (Cas) technology, 182,183 the introduction of mutations into the mouse or rat genome, in particular point mutations that were characterized in in vitro structure-function analyses or are present in humans as small nucleotide polymorphisms (SNPs), 11 opens the path to novel insights into the analysis of ECS components in the context of the entire organism.…”