SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors

Abstract

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3′UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans. Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3′UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

Mitochondria-Derived H2O2 Promotes Symmetry Breaking of the C. elegans Zygote

“…All constructs made in this study use the fbf-1 or mex-5 promoter and tbb-2 3'UTR region to achieve expression in the entire germline (Merritt et al, 2008). Fusing the individual fragments to generate the final genetic constructs was done using fusion PCR (Hobert, 2002) or SapTrap cloning (Fan et al, 2019). The engineered transgenes were cloned into pCFJ150 or pXF87 (Fan et al, 2019), sequence verified and integrated using the MosSCI method to create single-copy transgenes (Frøkjaer-Jensen et al, 2008) into strain EG6699, EG8078, EG8079, EG8080 or EG8082.…”

Modulating organelle distribution using light-inducible heterodimerization in C. elegans

“…To this end, LOVpep is tagged to the least dynamic structure of the two. For example, to transport mitochondria along microtubules we fused LOVpep to the relatively immobile mitochondria using TOMM-20 and fused dynein heavy chain DHC-1 to ePDZ ( Figure 1 C) ( Fan et al., 2019 ). On the other hand, to trap mitochondria at the cell membrane we fused LOVpep to a transmembrane domain to localize it to the plasma membrane, while ePDZ was now fused to the relatively more mobile mitochondria using TOMM-20 ( Figure 1 B) ( De Henau et al., 2020 ).…”

“…We successfully used mex-5 promoter or fbf-1 promoter in combination with tbb-2 3′UTR (sequences and expression patterns detailed in Merritt et al. (2008) ) to achieve LOVpep and ePDZ germline expression that is compatible with light-depending activation ( De Henau et al., 2020 ; Fan et al., 2019 ). Once the transgene is designed, codon optimization for expression in C. elegans ( Redemann et al., 2011 ), or the model organism of choice, is advised.…”

“…With the above in mind, we recommend the recently developed Golden Gate cloning technique SapTrap to assemble C. elegans MosSCI transgene vectors ( Fan et al., 2019 ) carrying LOVpep and ePDZ constructs in a fast, efficient, inexpensive, and scar-free manner ( Figure 2 ). The MosSCI backbone and donor plasmids carrying LOVpep and ePDZ that are compatible with this method are available at Addgene ( Fan et al., 2019 ). In the following we detail how MosSCI transgene vectors using the SapTrap method can be generated.…”

“…By designing SapI restriction fragments with complementary overhangs, multiple fragments can be assembled together in a defined order in a single digestion and ligation reaction ( Figure 2 B, the defined order is illustrated by the fragments ranging from light gray to dark gray). Primer design and examples of primers can be found in Figure 2 C and in the study by Fan et al. (2019) .…”