1 Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, USA
Workshop Time: 2:30pm- 5:30pm
Workshop Location: Sunset Village Grand Horizon Ballroom
Notification of Acceptance: May 19, 2017
We have recently used GRASP to label a substantial number of synapses in C. elegans (Oren-Suissa et al., 2016)(E.A.B., M.H., M.M., A.B. and O.H., unpubl. data) and want to share a few lessons that we learned:
1) To best visualize the area of contact between the two cells of choice, the neuronal processes should be labeled with a fluorescent cytoplasmic marker. Using both cellular markers in the same color is an option as long as the area of contact is clearly visible. Optimally, promoters should differ from those driving the split GFP fragments. The cell-specificity is less of a problem and promoters for cytoplasmic labeling could be expressed in additional cells.
2) We perform germ line transformation by microinjection. In cases where the GRASP signal is expected in ventral or dorsal areas of the body (for example, the PAG), we prefer to use the pRF4 (rol-6(su1006)) plasmid as the co-injection marker. It allows for easy visualization of GRASP puncta, as the roller worm is slightly rotated when mounted on a slide for imaging purposes. We co-inject 5 plasmids, two split GFP plasmids, two cytoplasmic markers and a co-injection marker. Alternatively, the GRASP construct could be injected into an already integrated cytoplasmic marker strain, so the marker and GRASP do not need to be on the same array.
3) Initial split GFP concentrations should be minimal and aimed at 10 ng/mcL. If same promoters are used for the cytoplasmic markers, the latter should be injected in extremely low concentrations, to avoid competition for transcription resources. All the heritable F2 worms are maintained for initial live-imaging analysis. As many transgenic lines as possible should be screened to uncover ones with synaptic GFP puncta localization. Imaging should be carried out as described below. Positive transgenic lines are those with discrete GFP puncta between the two cells. If expression is too high, synapses are saturated with GFP and the split-GFP interaction is irreversible. Transgenic animals generated by microinjection carry large extrachromosomal arrays incorporating a high copy number of transgenes, which might cause overexpression and germ line silencing. In addition, the inheritance of extrachromosomal arrays is unstable and can create genetic mosaics. We integrate the extrachromosomal arrays into C. elegans chromosomes for stable inheritance, using γ-irradiation. In some cases integrated GRASP strains underwent silencing. Growing these strains at 25C for a couple generations disilenced these arrays.
4) Transgenics lines with good signals are often hard to find. Concentration of split-GFP plasmids should be gradually increased, either of both plasmids or of one of them. A common solution is to swap the split GFP fragment between the pre- and the post-synaptic cells. Alternatively, different neuronal promoters should be tried.
5) GRASP imaging conditions: Compared to our Zeiss Imager Z1 standard compound epifluorescence microscope, we have observed much better signals on a pinhole based confocal microscope (LSM880). Epi-fluorescent microscope images contain too much out-of-focus signals. Puncta were quantified by scanning the original full Z-stack for distinct dots in the area where the processes of the two neurons overlap.
Feinberg, E.H., Vanhoven, M.K., Bendesky, A., Wang, G., Fetter, R.D., Shen, K., Bargmann, C.I., 2008. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353-363.
A readily available fosmid library covers ~90% of all worm genes. We have previously described a pipeline for manipulating the sequence of fosmid clones via a recombineering strategy in E.coli (Tursun et al., 2009, PLoS One. 2009;4(3):e4625). Recombineered fosmids permit expression of fluorescent reporters or genetically altered protein products, in transgenic worms, under the control of most, if not all, endogenous regulatory sequences of the gene of interest. We report here more than a dozen new cassettes for fosmid recombineering using our pipeline. These cassettes allow for a number of different types of tagging for biochemical and microscopy purposes among others, all listed in Table 1.
In light of the advent of CRISPR/Cas9-mediated genome engineering, it is important to point out the persistent value of fosmid-based transgenesis: (a) Single copy fluorescent proteins generated by CRISPR/Cas9 or miniMos can be difficult to visualize, especially if the gene has low expression. The multicopy nature of transgenic arrays can increase the intensity of the reporter gene signal to a detectable level. (b) Some genes remain difficult to target by CRISPR, either for technical or biological reasons (e.g. adding a tag generates a hypomorphic allele that might not provide enough gene function in single copy). In our hands, fosmid recombineering in bacteria is fast and always successful. Finally, (c) fosmid-based reporter transgenes provide a fast and straightforward tool for both rescue of mutant phenotypes and subsequent mosaic analysis.
The complete toolkit can be requested from the Hobert lab (firstname.lastname@example.org).
If possible, we use the native CRISPR/Cas9 system, which relies on Cas9 protein with both a universal RNA (tracrRNA) and a specific guide RNA (crRNA). These RNAs are bought from Dharmacon, and the same tracrRNA can be used for every injection. Although commercial Cas9 is also available, it is expensive. Thus, Paix and Seydoux developed a protocol for Cas9 production in E. coli . We modified their procedure to simplify the Cas9 purification. Briefly, the plasmid NM2973 was transformed into NEB Nico-21 competent cells (#C2529H), and Cas9 expression was induced by adding IPTG to the culture media. We purified Cas9 under native conditions, using a Qiagen Fast-Start Kit (#30600). Following instructions, the bacterial pellet was resuspended in 10 ml of lysis buffer, and cooled on ice for 30 minutes. The cell lysate supernatant flowed through the Fast-Start Ni-NTA column by gravity, and 4 ml of washing buffer was used to wash the column twice. Next, Cas9 was eluted with 2 ml of elution buffer. Finally, the Cas9 eluate was centrifuged with a centricon filter (#UFC905024) to concentrate the protein. We obtained 2.5 mg of Cas9 from 1L of culture, and made 2 µl aliquots at a concentration of 10 µg/µl for storage at -80°C.
Home-made Cas9 was tested to measure it efficiency. First, we performed a cleavage assay to confirm that it had endonuclease activity. A mixture of Cas9, mouse SLC guide RNA and a PCR fragment from the SLC gene were digested at 37°C for 1 hour. The result showed that our Cas9 completely cleaved the SLC fragment as shown in Figure 1. Second, we used the Cas9 to knockout the cbr-dpy-5 gene in vivo. The synthetic cbr-dpy-5 crRNA and tracrRNA were incubated with Cas9 at 37°C for 15 minutes, and microinjected into the gonads of C. briggase hermaphrodites. Among all the F1 progeny, 25% produced homozygous Dpy children. A similar injection using commercial Cas9 (PNA Bio.) gave 18% affected F1s. Sequencing confirmed that the cbr-dpy-5 mutation was a 51 bp deletion near the PAM site of its target. Thus, home-made Cas9 has high efficiency and allows rapid and inexpensive gene editing.
If the desired target lacks a nearby 3’-GGnGG, TALENs can be used for precise gene editing. Since the only constraint for TALEN binding is that the first nucleotide in each target sequence is a T, sites exist almost everywhere in the genome. The plasmids to synthesize a pair of TALEN mRNAs can easily be built in a week, using a procedure developed by Cermak et al , with the destination vector pRE189 for expressing nematode mRNAs . A Golden Gate TALEN kit is available from Addgene (#1000000024) and mRNA kits from Ambion (#AM1344, #AM1908).
Acknowledgments: We thank Dr. Geraldine Seydoux for the NM2973 plasmid. This work was supported by NIH grant to Dr. Ronald E Ellis.
 Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, and Voytas DF. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39(12):1-11
 Paix A, Kolkmann A, Rasoloson D, and Seydoux G. (2015) High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics. 201(1):47-54
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