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Setting up simple and cost-effective gene editing for nematodes

Yongquan Shen1 and Ronald E Ellis1
1 Department of Molecular Biology, Rowan University-SOM, Stratford, NJ 08084
Correspondence to: Ronald E Ellis PhD (ellisre@rowan.edu)
Genome editing with CRISPR/Cas9 or TALENs has become an essential tool for working with nematodes [3,4,5]. Plasmids expressing Cas9 and a custom sgRNA can be used for gene editing in many situations, particularly for C. elegans [2]. However, this approach is less effective for C.briggase and some male-female species. Furthermore, some edits will be difficult if a good Cas9 target is not located nearby.Our approach is to use Cas9 protein complexes for injections [4] when Cas9 target sites are available, and TALENs for other situations [5]. The best targets for Cas9 end in a 3’-GGnGG sequence [3], which is infrequent in worms. Although satisfactory sites can usually be found for gene knockouts, they might not be available for site-specific editing.

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 [4]. 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 [1], with the destination vector pRE189 for expressing nematode mRNAs [5]. 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.

Figures



Figure 1: Cas9 Cleavage assay in vitro

References

[1] 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  PubMed

[2] Dickinson DJ, And Goldstein B. (2016) CRISPR-based methods for Caenorhabditis elegans genome engineering. Genetics. 202(3):885-901  PubMed

[3] Farbound B, and MeyerBJ. (2015) Dramatic enhancement of genome editing by CRISPR-Cas9 through improved guide RNA design. Genetics. 199(4):959-971  PubMed

[4] 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  PubMed

[5] Wei Q, Shen Y, Chen X. Shifman Y. and Ellis RE. (2014) Rapid creation of forward genetics tools for C. briggase using TALENs: lessons from nonmodel organisms. Mol. Biol. Evol. 31(2):468-473  PubMed

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Analysis of mitochondrial sodium, magnesium, calcium, manganese, and iron in wild-type (N2) C. elegans

Laura L. Maurer1, Chuanjia Jiang2, Heileen Hsu-Kim2, Joel N. Meyer1
1 Nicholas School of the Environment, Duke University, Durham NC
2 Department of Civil & Environmental Engineering, Duke University, Durham NC
Correspondence to: Laura L. Maurer (laura.kubik@duke.edu)
Mitochondria are dynamic organelles that require a controlled balance of essential ions to perform critical functions within cells. The content of these elements can both influence the effect of and be influenced by different stressors, including aging (Klang et al., 2014) and exposure to toxic chemicals (Sahu et al., 2013). This article provides reference concentrations of five essential ions present in the mitochondrial fraction of young adult N2 C. elegans. Briefly, 50,000 N2s were grown to young adult stage for mitochondrial isolation. Mitochondria were isolated from whole nematodes by dounce homogenization and differential centrifugation into a crude mitochondrial fraction as described (Kayser et al., 2004) with minor modifications. Elemental analysis of mitochondrial fractions was performed by inductively coupled plasma mass spectrometry (ICP-MS). Samples were prepared by oven drying (95°C overnight) and HNO3 digestion (90°C for 4 h), followed by dilution with MilliQ water to a final HNO3 concentration of 3% prior to elemental analysis by ICP-MS. Results were normalized to protein content (BCA assay). Sodium (Na) was present in the highest concentrations compared to other elements (significantly higher than calcium (Ca), magnesium (Mg), iron (Fe), and manganese (Mn); One-way ANOVA with Tukey’s post-hoc test, *p≤0.05; means are denoted above each bar). Mitochondrial Na concentrations are significantly higher than Ca concentrations in mammalian cells (Murphy and Eisner, 2009), and mitochondria can be a sink for magnesium (Kubota et al., 2005) as well as iron in mammals. It should be noted, however, that measured mitochondrial ion concentrations are influenced greatly by the method used for isolation; caution should be exercised when interpreting relative ion concentrations. These data serve as reference concentrations for mitochondria isolated from young adult N2 utilizing the cited methods.

Figures


ng_ug protein Mito Fraction N2

References

Kayser, E.B., Morgan, P.G., and Sedensky, M.M. (2004). Mitochondrial complex I function affects halothane sensitivity in Caenorhabditis elegans. Anesthesiology 101, 365-372.  PubMed

Klang, I.M., Schilling, B., Sorensen, D.J., Sahu, A.K., Kapahi, P., Andersen, J.K., Swoboda, P., Killilea, D.W., Gibson, B.W., and Lithgow, G.J. (2014). Iron promotes protein insolubility and aging in C. elegans. Aging (Albany NY) 6, 975-991.  PubMed

Kubota, T., Shindo, Y., Tokuno, K., Komatsu, H., Ogawa, H., Kudo, S., Kitamura, Y., Suzuki, K., and Oka, K. (2005). Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells. Biochim Biophys Acta 1744, 19-28.  PubMed

Murphy, E., and Eisner, D.A. (2009). Regulation of intracellular and mitochondrial sodium in health and disease. Circ Res 104, 292-303.  PubMed

Sahu, S.N., Lewis, J., Patel, I., Bozdag, S., Lee, J.H., Sprando, R., and Cinar, H.N. (2013). Genomic analysis of stress response against arsenic in Caenorhabditis elegans. PLoS One 8, e66431.  PubMed

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Silver nanoparticles are in general more toxic to C. elegans than tested gold, copper, iron, titanium dioxide, zinc oxide, cerium oxide, and carbon-based nanoparticles

Joel N. Meyer, Alex H. Simon, Krithika Umakanth, Xinyu Yang
Nicholas School of the Environment, Duke University, Durham NC, USA
Correspondence to: Joel Meyer (jnm4@duke.edu)
Commercial use of nanoparticles (NPs) is increasing rapidly. There is concern that their unique physicochemical properties could cause toxicity, but testing is complicated by the fact that there are a very large number of NPs being invented, with varied materials, sizes, shapes, and surface modifications. To save others the potential effort of replicating toxicologically uninteresting results, we report that with the exception of silver (Ag) NPs, most that we have tested have been essentially nontoxic to C. elegans under laboratory conditions except at very high concentrations. We compare the toxicity of NPs that are only toxic at high concentrations to values for AgNPs that we have published on (Yang et al. 2012; Arnold et al. 2013), because they are more toxic. Toxicity was tested either as inhibition of larval growth, or survival of young adults. Exposures were carried out in moderately hard reconstituted (“EPA”) water, as described (Maurer et al. 2015). We present NPs rank-ordered for toxicity (Table 1; most toxic at top), based on inhibition of growth of L1 larvae over 72 h. EC50 (concentration that reduced growth by 50%) values, with one exception, were 10-1000x lower for AgNPs than other NPs. We found the same general result when assessing young adult survival (young adults are generally more resistant to NPs than larvae (Zhao et al. 2013; Choi et al. 2014)), with some of the same and some different NPs. 24 h LC50 (concentration that kills 50%) values were: uncoated and polyvinylpyrrolidone-coated Ag NPs (sizes in Table 1), between 0.04 and 3.3 ppm; 34 nm citrate-tannic acid-coated AgNPs, 3.5 ppm; 29 nm citrate-glutamate-coated AgNPs, 1.4 ppm; 40 nm CuONPs, 10-30 nm ZnONPs, 20-30 nm FeONPs, 15 nm citrate-coated AuNPs, 10 x 40 nm rutile TiO2NPs, and 10-30 nm rutile TiO2NPs, > 100 ppm. This is not a comprehensive analysis of NP toxicity. For example, environmental conditions can alter NP toxicity: sunlight can greatly increase, and sulfidation, organic matter and other factors decrease, toxicity (Maurer et al. 2015; Zhao et al. 2013; Choi et al. 2014). It is also possible that longer-term and subtler health impacts of NPs, or effects on organs absent in C. elegans (e.g., lungs), will be important.

Tables

WBG NPs table

References

Yang X, Gondikas AP, Marinakos SM, Auffan M, Liu J, Hsu-Kim H, et al. (2012). Mechanism of silver nanoparticle toxicity is dependent on dissolved silver and surface coating in Caenorhabditis elegans. Environ. Sci. Technol. 46, 1119-1127.  PubMed

Arnold MC, Badireddy AR, Wiesner MR, Di Giulio RT, Meyer JN. (2013). Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans. Arch. Environ. Contam. Toxicol. 65, 224-233.  PubMed

Maurer LL, Ryde IT, Yang X, Meyer JN. (2015). Caenorhabditis elegans as a Model for Toxic Effects of Nanoparticles: Lethality, Growth, and Reproduction. Curr. Protoc. Toxicol. 66, 20 10 21-20 10 25.  PubMed

Zhao YL, Wu QL, Li YP, Wang DY. (2013). Translocation, transfer, and in vivo safety evaluation of engineered nanomaterials in the non-mammalian alternative toxicity assay model of nematode Caenorhabditis elegans. Rsc. Adv. 3, 5741-5757.  PubMed

Choi J, Tsyusko OV, Unrine JM, Chatterjee N, Ahn JM, Yang XY, Thornton BL, Ryde IT, Starnes D, Meyer JN. (2014). A micro-sized model for the in vivo study of nanoparticle toxicity: what has Caenorhabditis elegans taught us? Environ. Chem. 11, 227-246.  PubMed

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Fast and inexpensive C. elegans genomic DNA preparation using Sigma’s Extract-N-Amp kit

Bhoomi Madhu1 and Tina L. Gumienny1
1Department of Biology, Texas Woman’s University, Denton, Texas, USA
Correspondence to: Tina L. Gumienny (tgumienny@twu.edu)
PCR of a single or a few nematodes is a standard protocol for many C. elegans laboratories. The standard method to prepare genomic DNA template for PCR is to freeze animals in worm lysis buffer containing Proteinase K in an ultralow freezer (-70°C – 80°C) for at least 15 to 45 minutes (longer is recommended by some protocols) (Williams et al., 1992, Fay and Bender, 2008, Biron and Haspel, 2015). This is followed by a one-hour incubation at 60°C – 65°C for the Proteinase K to work, followed by 15-30 minutes at 95°C to inactivate the enzyme activity.We have adapted the Sigma Extract-N Amp kit for use with C. elegans (XNAT2 kit, http://www.sigmaaldrich.com/catalog/product/sigma/xnat2?lang=en&region=US).  Although the instructions in the kit provide directions for preparing C. elegans DNA, using “1 C. elegans in solution” in 225 µL total solution volume, we modified this kit protocol to prepare single animals for PCR analysis and to genotype lines using PCR (Weber and Douglas, 2016). Our method 1) decreases the template preparation time to 15 minutes, 2) decreases the total volume needed to extract the DNA from one animal over 100-fold from the volume specified in the kit instructions, and 3) is at least two times less expensive than the Proteinase K method. Although the cost depends on the amount ordered and the supplier, our method using the Sigma kit costs about $0.007 per reaction, whereas Proteinase K alone costs approximately $0.015- $0.02 per reaction.

Protocol 1: Single worm DNA extraction using the Sigma Extract-N-Amp kit

All steps are done at room temperature.

Start a thermocycler program to heat to 55°C (hold), 55°C for 10 minutes, then 95°C for 3 minutes.

Aliquot 0.8 µL Extraction Solution into a PCR tube. Add 0.2 µL Tissue Preparation Solution. Mix by pipetting.

Pick 1 gravid adult animal into the solution.

Centrifuge the tube briefly (2-3 seconds, ≤ 6000 rpm/2,000xg).

Put the tube in the thermocycler and continue the program (55°C for 10 minutes, 95°C for 3 minutes). When the program is complete, centrifuge the tube briefly.

Add 0.8 µL Neutralization Solution to the mixture and mix by pipetting. The extract can be used for PCR directly or can be stored at 4°C for future use.

Samples will be ready from worm pick to PCR within 15 minutes.

 

Protocol 2: DNA extraction of a few individuals

This method is useful for genotyping crosses, for example, or if a little more than a single animal’s DNA is needed. This yields a final concentration of 3.5 nematodes/µL. 1 µL of this template usually works for our 25 µL PCRs, but optimization of template concentration may be required. This protocol yields enough template (at 1 µL/reaction) for four reactions. With 16 animals’ genomic DNA mixed together, this template is certain to accurately genotype a line (barring fickle primers that preferentially amplify a smaller band or other technical issues).

All steps are done at room temperature.

Start a thermocycler program to heat to 55°C (hold), 55°C for 10 minutes, then 95°C for 3 minutes.

Aliquot 2.0 µL Extraction Solution in a PCR tube. Add 0.5 µL Tissue Preparation Solution to it. Mix the solutions by pipetting.

Pick 16 gravid adult animals into the solution in a PCR tube.

Centrifuge the tube briefly (2-3 seconds, ≤ 6000 rpm/2,000xg).

Put the tube in the thermocycler and continue the program (55°C for 10 minutes, 95°C for 3 minutes). When the program is complete, centrifuge the tube briefly.

Add 2.0 µL Neutralization Solution to the mixture and mix by pipetting. The extract can be used for PCR directly or can be stored at 4°C for future use.

Samples are ready for PCR within 15 minutes.

We found that SapphireAmp Fast PCR Master Mix (Takara Bio USA, Inc, Mountain View, CA, USA) further reduces total time and works more reliably than the PCR Reaction Mix provided in the Extract-N-Amp XNAT2 kit.

References

Biron, D., and Haspel G. (2015). C. elegans Methods and Applications. Methods in Molecular Biology 1327, 1-10.  PubMed

Fay, D. and Bender, A. SNPs: Introduction and two-point mapping (September 25, 2008), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.93.2.  PubMed

Weber, S., and Douglas, D. (2016). Extract-N-Amp Tissue Feature Article. Sigma-Aldrich Corporation, St. Louis, MO, USA. http://www.sigmaaldrich.com/technical-documents/articles/biology/extract-n-amp-tissue-feature-article.html  PubMed

Williams, B.D., Schrank, B., Huynh, C., Shownkeen, R., and Waterston, R.H. (1992). A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. Genetics 131, 609-624.  PubMed

  PubMed

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Brief report on the use of the phiC31 integrase for the transgenic array engineering

Wadim Kapulkin1,*, Andrei Pozniakovsky1
1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
* present affiliation: Veterinary Consultancy, Conrada 16/65, 01-922 Warsaw, Poland
Correspondence to: Wadim Kapulkin (wadim_kapulkin@yahoo.co.uk)
In systems other than worms (i.e. and also vertebrate cells) Streptomyces temperate phage phiC31 integrase was reported as an efficient mean of inserting the transgenes into specific sites of the genome. In the summer of 2013 we have made an experimental attempt to evaluate if phiC31 integrase would be of possible use in C. elegans. For this purpose we implemented the unc-119(ed3) mutant rescue (1), by linking of two split segments of the unc-119 rescuing cassette. Split segments of unc-119 are two separate plasmids providing respectively: unc-119 promoter and N-terminal UNC-119 CDS and C-terminal UNC-119 followed by unc-119 3′ utr (split at the third intron and flanked with the appropriate phiC31 recognition sites). In addition the third construct was used, intended to provide the phiC31 integrase into the germline cells (essentially based on pJL43.1 – p-glh-2::MosTase::glh-2-utr, where the Mos1 transposase (2, 3) was replaced by codon optimized phiC31 integrase) to facilitate the transgene engineering.

Using the above constructs, we obtained the following evidence for the phiC31 acting efficiently on the transgenic extrachromosomal arrays. We assumed here, the two separate plasmids containing split of two segments of unc-119 gene will rescue the ed3 allele in DP38 derived strain, only if properly re-assembled into functional unc-119 gene. To test for the above assumption the above plasmids, where co-injected into unc-119(-) hermaphrodites carrying homozygous ed3 allele. In the first experiment, two of the above plasmids containing split of two segments of the unc-119 gene, were injected (along with fluorescent markers*) into gonads of the DP38 hermaphrodites. Based on the segregation of the fluorescent markers in the transgenic progeny in this experiment, we concluded that while the injected split unc-119 segments do efficiently form the heritable transgenic array, however fail to rescue the uncoordinated phenotype (observed at least four 4 independent array forming events in 30 injected hermaphrodites).

We contrast the above observation, with the results of the second experiment, where the above plasmids where co-injected with the phiC31 integrase providing plasmid (again along with fluorescent markers) into gonads of DP38 derived hermaphrodites. We observed at least 4 independent events (in 28 injected hermaphrodites) of formation of the heritable arrays resulting in non-uncoordinated movement in unc-119(-) animals, when the split segments of unc-119 were provided with the third plasmid encoding for phiC31 integrase. Consistently the above four of the unc-119 rescued lines, were proven resistant to G418 indicating for the transmission of the selectable marker on the extrachromosomal array and the restored unc-119 function, depending strictly on the germinal provision of the phiC31 expressing plasmid. Collectively, we think we do have  enough evidence, to conclude the phiC31 is working on the transgenic arrays formed of the split unc-119 segments. We think this demonstration could be possibly useful in for example array-array transgenic engineering technology i.e. phiC31 targeted  linking of the two separate extrachromosomal arrays.

* those lines where propagated under G418 selection, as the intact NeoR gene was included into one of the constructs, providing additional line of the confidence over the selection based on fluorescent markers when maintaining the transgenic arrays in the (slow growing) non-rescued uncoordinated unc-119(-) line.

Acknowledgments: We are grateful to Mihail Sarov (www.mpi-cbg.de/services-facilities/core-facilities/genome-engineering-facility/), for providing the p-glh-2 driven phiC31 integrase construct and the friendly and supportive laboratory environment.

References

Maduro M, Pilgrim D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics. 1995 Nov;141(3):977-88.   PubMed

Bessereau JL, Wright A, Williams DC, Schuske K, Davis MW, Jorgensen EM. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature. 2001 Sep 6;413(6851):70-4.
  PubMed

Frøkjaer-Jensen C, Davis MW, Hopkins CE, Newman BJ, Thummel JM, Olesen SP, Grunnet M, Jorgensen EM. Single-copy insertion of transgenes in Caenorhabditis elegans
Nat Genet. 2008 Nov;40(11):1375-83. doi: 10.1038/ng.248.
  PubMed

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