Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

Abstract

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 C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans 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 C. elegans: 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. However, 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 C. elegans 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. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

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) (Table S2). 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, 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 Caenorhabditis 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 PPIs that can utilize many existing strains and produces a clear result with commonly available equipment in any C. elegans laboratory.…”

Germline TFAM levels regulate mitochondrial DNA copy number and mutant heteroplasmy inC. elegans

“…To overexpress TFAM (encoded by the hmg-5 gene) in the germ line, we used regulatory elements from the endogenous glh-1 gene, which encodes a highly expressed germline-specific protein (Marnik et al 2019; Goudeau et al 2021). We employed two strategies to express untagged TFAM at the glh-1 locus: the viral 2A self-cleaving peptide system, which results in the production of two peptides via ribosomal skipping during translation ( Fig.…”