Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent labeling of endogenous proteins inCaenorhabditis elegans

Abstract

<jats:title>Abstract</jats:title><jats:p>We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous <jats:italic>C. elegans</jats:italic> proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of <jats:italic>C. elegans</jats:italic> proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in <jats:italic>C. elegans</jats:italic>: the small fragment can quickly and easily be fused to almost any protein of interest and can be detected wherever the large fragment is expressed and complemented. There is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available <jats:italic>C. elegans</jats:italic> lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet, and generate transgenic <jats:italic>C. elegans</jats:italic> lines to allow easy single-color labeling in muscles and dual-color labeling in somatic cells. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for an easy, cloning-free method for CRISPR/Cas9 editing.</jats:p>

A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage

“…To visualize age-related protein aggregation, we monitored the casein kinase subunit KIN-19, which forms insoluble aggregates in an age-dependent manner in wild-type somatic tissues ( David et al, 2010 ). To do this, we fluorescently tagged the endogenous kin-19 gene specifically in the soma using split-wrmScarlet ( Goudeau et al, 2021 ; Supplementary file 2 ). To avoid interference from the bright intestinal autofluorescence, we examined KIN-19::split-wrmScarlet in the head.…”

“…Split-wrmScarlet 11 was introduced at the C-terminus of KIN-19 using CRISPR/Cas9 and single-stranded oligodeoxynucleotide template (ssODN) in the strain CF4582 expressing split-wrmScarlet 1-10 in somatic tissues (driven by the eft-3 promoter and unc-54 3′UTR) to obtain the strain CF4609. CRISPR insertion of split-wrmScarlet 11 was performed following published protocols ( Goudeau et al, 2021 ; Paix et al, 2016 ; Paix et al, 2015 ). Briefly, ribonucleoprotein complexes (protein Cas9, tracrRNA, crRNA) and ssODN were microinjected into the gonads of young adults using standard methods ( Evans, 2006 ).…”

A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage

“…To visualize age-related protein aggregation, we monitored the casein kinase subunit KIN-19, which forms insoluble aggregates in an age-dependent manner in wild-type somatic tissues (David et al, 2010). To do this, we fluorescently-tagged the endogenous kin-19 gene specifically in the soma using split-wrmScarlet (Goudeau et al, 2020a) (Table S2). To avoid interference from the bright intestinal autofluorescence, we examined KIN-19::wrmScarlet in the head.…”

“…WrmScarlet 11 was introduced at the C-terminus of KIN-19 using CRISPR/Cas9 and single-stranded oligodeoxynucleotide template (ssODN) in the strain CF4582 expressing wrmScarlet 1-10 in somatic tissues (driven by the eft-3 promoter and unc-54 3’UTR), to obtain the strain CF4609. CRISPR insertion of wrmScarlet 11 was performed following published protocols (Goudeau et al, 2020a; Paix et al, 2016, 2015). Briefly, ribonucleoprotein complexes (protein Cas9, tracrRNA, crRNA) and ssODN were microinjected into the gonad of young adults using standard methods (Evans, 2006).…”

CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

“…While we mainly use proteins tagged with mCherry, proteins tagged with YFP, mScarlet, mKate2, or BFP could be used with the system. With the recent development of CRISPR/Cas9 editing techniques and split protein fluorescent based systems, such as sfGFP 11 and Split-mScarlet 11 (Goudeau et al, 2021), where only a small fragment of the fluorescent protein needs to be integrated in a genetic background expressing the complementary fragment, fluorescent protein tags can be made with relative ease. Overall, the CeLINC system is a powerful technique to study protein-protein interactions that can utilize many existing strains and produces a clear result with commonly available equipment in any C. elegans laboratory.…”