The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.

1. Construction of the LED Illuminator
NOTE: The required LED equipment is summarized in the List of Materials. The entire LED setup is small and can be placed anywhere in the lab, although we recommend it be placed in a dark room to control light exposure of worms4.
<ol>
	<li>Connect the LED to a controller with cables (Figure&#160;1A,&#160;D).</li>
	<li>Connect the controller to a digital function generator/amplifier with a BNC cable (Figure 1A, C, D).</li>
	<li>Fix the LED light 10 cm above the stage using a custom holder (Figure 1A). 
	NOTE: We made the holder with metal parts.</li>
	<li>Cover the LED illumination setup partially, but avoid heat accumulation (Figure 1B), which makes worms sick. 
	NOTE: We use a custom-made hard plastic cover with open top and bottom (Figure 1A). The cover is not required for the mutagenesis itself, but is used to limit unnecessary exposure of the blue light to the surroundings. We advise to not cover the LED setup entirely (Figure 1B) to prevent overheating the worms.</li>
	<li>Wear laser-protective glasses. 
	NOTE: We wear laser-protective glasses because the light is very bright. Sunglasses may work as well.</li>
	<li>Switch the lever of the LED controller to &#39;Int.&#39;&#160;for continuous illumination (Figure 1D) and set it at 65% of the maximum power (Figure 1C).</li>
	<li>Use a photometer to measure the blue light intensity on the stage where the worms are placed using manufacturer&#39;s protocol. The setup gives 2.0 mW/mm2 under continuous illumination.</li>
</ol>
2. Blue Light Treatment
NOTE: Use the strain CZ20310 juSi164[Pmex-5-HIS-72::miniSOG-3&#39;UTR(tbb-2) + Cb-unc-119(+)] unc-119(ed3)III4. The strain is available at the Caenorhabditis Genetics Center (CGC, http://www.cgc.cbs.umn.edu/).
<ol>
	<li>Maintain the His-mSOG strain in the dark on standard nematode growth medium (NGM) plates with E. coli OP50 following a standard protocol10,11. 
	NOTE: Under ambient light, juSi164-containing strains do not display a mutation rate above a spontaneous rate4. Routine strain passage using a dissecting scope with a halogen lamp generally does not cause mutations as well. Nonetheless, care should be taken to avoid unnecessary exposure to light, e.g., keeping the strains in a standard dark-inside incubator, or covering the strains with foil. It is advised to freeze aliquots of the strain after receiving it in case unnecessary mutations accumulate over time.</li>
	<li>Pick gravid young adult (approximately 12 h&#160;post-L4) animals with a worm pick11. 
	NOTE: Younger animals show less mutagenic effect4, while older animals may have lower brood size.</li>
	<li>Cut a filter paper to fit a 60 mm plate with a 25 mm x 25 mm square hole and place onto an unseeded plate (Figure 1E). 
	NOTE: A square punch can be used but the&#160;size of the punch does not have to be precise.</li>
	<li>Drop 100 &#181;L of 100 mM CuCl2 on the filter paper evenly to restrict worms within the square hole on the plate.</li>
	<li>Pick desired number of gravid young adult hermaphrodites (see Step 3 for an example) and transfer to the center of the CuCl2 plate.</li>
	<li>Wear laser protective glasses.</li>
	<li>Switch ON the LED and the function generator.</li>
	<li>Switch the lever of the LED controller to &#39;Ext.&#39;&#160;to control it by the function generator (Figure 1D).</li>
	<li>Set the controller at 65% of the maximum power and the function generator as sine wave, 4 Hz (Figure 1C).</li>
	<li>Place the CuCl2 plate from Step 2.5 without a lid on the stage under the LED (Figure 1A).</li>
	<li>Illuminate the animals with blue light for 30 min.</li>
	<li>Transfer healthy looking worms to a seeded plate as P0. 
	NOTE: The desired number of P0 depends on the type of screen. See Step 3 for an example. The P0 worms may appear to be uncoordinated immediately after light treatment and will recover over time.</li>
	<li>Keep worms covered using foil in an incubator or in the dark; and follow the standard procedure to begin screening desired phenotypes among their progeny2,10.</li>
</ol>
3. Example of a Forward Genetic Screen
NOTE: This is an example of the clonal screen with 120 haploid genomes to check if the LED and strain are working. Figure 2 shows a schematic of the procedure.
<ol>
	<li>[Day1] After blue light treatment, pick five healthy looking worms onto a NGM plate with OP50 as P0, keep them in a 20 &#176;C incubator, and let them lay eggs for 1 d (&#39;Day1&#39; plate).</li>
	<li>[Day2] Transfer P0 onto a new seeded plate (&#39;Day2&#39; plate).</li>
	<li>[Day3] Pick and kill P0 on the &#39;Day2&#39; plate by burning them.</li>
	<li>[Day4] Pick 30 gravid young adult F1 from the &#39;Day1&#39; plate and transfer them onto individual plates (30 F1 plates for &#39;Day1&#39;).</li>
	<li>[Day5] Repeat Step 3.4 for the &#39;Day2&#39; plate (30 F1 plates for &#39;Day2&#39;).</li>
	<li>[Day7-9] Check the abnormal morphologies and/or behavior of F2 worms compared to the WT strain (Figure 3A) under a standard stereomicroscope when they become adults. 
	NOTE: A heritable mutation displays about a quarter of worms with the same phenotype among F2 when it is recessive with high penetrance. However, some mutants may grow slowly and/or show partial penetrance. Therefore, it is possible that less than a quarter of the mutants can be found on a plate.</li>
	<li>Pick worms with visible phenotypes individually and check the heritability of the phenotype in the next generation.</li>
</ol>
4. Example of an Extrachromosomal Transgene Integration
<ol>
	<li>Inject the DNA of interest into CZ20310 juSi164[Pmex-5-HIS-72::miniSOG-3&#39;UTR(tbb-2) + Cb-unc-119(+)] unc-119(ed3) and prepare transgenic worms with a low transmission rate (10-30%) following a standard injection protocol12,13. 
	NOTE: Alternatively, established extrachromosomal arrays can be crossed into the juSi164 strains. To avoid genotyping for juSi164, juSi164 heterozygotes may be used as the P0 although efficiency might be decreased. The genotyping method for juSi164 is described in Section 5.</li>
	<li>[Day1] Treat ~20 transgenic animals as P0 with blue light as described in Step 2. 
	NOTE: More P0 worms are necessary depending on the transmission rate. Transgenes can be identified by phenotypes or coinjection marker12,13.</li>
	<li>Pick 10 healthy looking P0 onto individual plates, keep them in a 20 &#176;C incubator, and let them lay eggs for a few days (P0 plates)</li>
	<li>[Day4] Single out 20 transgenic F1 worms from each P0 plate onto individual plates and let them lay eggs. (20 F1 x 10 P0 plates= 200 F1 plates in total.)</li>
	<li>[Day7] Find F1 plates with &#62;75% F2 having transgenes.</li>
	<li>Pick five transgenic animals from the &#62;75% transmission rate F1 plates (F2 plates).</li>
	<li>[Day10] Keep F2 plates with 100% transmission rate as potential integrated lines.</li>
	<li>Outcross the potential integrated lines using a standard procedure10 to check the Mendelian segregation and to remove potential background mutations.</li>
</ol>
5. Genotyping
<ol>
	<li>Lyse 20 outcrossed F2 worms in lysis buffer using a standard protocol14.</li>
	<li>Perform PCR using the primers for MosSCI insertion9, juSi164, (Table 1) with an annealing temperature of 58 &#176;C following a standard protocol14. 
	NOTE: It is unnecessary to genotype unc-119(ed3) because unc-119(ed3) can be detected by uncoordinated behavior after removing the rescuing transgene (juSi164) in the following Steps 5.4 and 5.5.</li>
	<li>Run agarose gel electrophoresis15 and examine the band sizes (Table 1) to find the WT&#160;for juSi164.</li>
	<li>Examine if the F2 progeny has paralyzed worms due to the existence of unc-119(ed3).</li>
	<li>If uncoordinated worms are found on step 5.4, single several nonuncoordinated worms to obtain all nonuncoordinated worms in the next generation.</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a detailed procedure of optogenetic mutagenesis using His-mSOG expressed in the germline and provide an example of a small scale forward genetic screen. Compared to standard chemical mutagenesis, this optogenetic mutagenesis method has several advantages. Firstly, it is nontoxic, thereby keeping lab personnel away from toxic chemical mutagens such as EMS, ENU and trimethylpsoralen with ultraviolet light (TMP/UV). Secondly, the experimental procedure of mutagenesis can be used for a small number of the P0 animals and can be finished within one hour. Thirdly, the optogenetic mutagenesis produces a broad spectrum of mutations including nucleotide substitutions&#160;and chromosome deletions. Finally, the optogenetic mutagenesis can be used for integration of extrachromosomal transgenes.
The critical step in this mutagenesis protocol is to ensure that the LED setup works on His-mSOG containing strains at the expected efficiency. We recommend performing a pilot clonal screen, as described in Protocol Section 3, with about 100 haploid genomes. The mutagenesis is easily scalable from the small scale pilot screens to nonclonal screens by increasing the number of P0 for the light treatment. For large-scale nonclonal screens, we suggest synchronizing the worms to gravid young adults and transferring hundreds of them with M9 solution onto a CuCl2 plate for light treatment. The standard condition described in Steps 2 and 3 is estimated to have approximately 4.4 times lower efficiency than the widely used 50 mM EMS mutagenesis method4. The mutation rate may be improved by increasing the light exposure time and/or increasing the intensity of the light. However, these modifications can cause phototoxicity and lead to small brood size and sickness of light-treated worms. To increase the efficiency, it may be possible to use miniSOG derivatives with higher ROS yield such as miniSOG(Q103L) although it may increase the background mutagenesis effect without light treatment16,17. For the modification of the DNA construct, the Pmex-5-HIS-72::miniSOG-3&#39;UTR(tbb-2) plasmid (pCZ886) is available commercially.
Under the conditions described here, integrated transgenes are generated at approximately one&#160;line per 200 F1 on average4 when using an extrachromosomal transgenic strain with a transmission rate of 10 - 30%. This efficiency may be increased by using extrachromosomal transgenic strains with high transmission rates as previously reported for a UV radiation method18.
His-mSOG-mediated optogenetic mutagenesis induces mutations in a wide range of genes. Also, the frequency of loci recovered from the rpm-1 suppressor screen using optogenetic mutagenesis is similar to those from the same screen using EMS4. However, it is possible that optogenetic mutagenesis using a histone gene may have a bias towards certain genes, due to unknown factors such as chromatin structure. To address this issue, it is necessary to analyze many optogenetically induced mutations by whole genome sequencing.
His-mSOG-mediated optogenetic mutagenesis causes a wide spectrum of mutations including single nucleotide substitutions and deletions ranging from 1 bp to a few hundred bp4. The spectrum of single nucleotide variants suggests that miniSOG-induced reactive oxygen species are the cause of the mutations4. It is also possible that miniSOG damages histone proteins and causes chromatin instability because miniSOG is used to inactivate proteins as well19. It may be possible to manipulate the type of mutations by using light of different intensities. Since optogenetic mutagenesis causes deletions, it is important to analyze deletions as well as single nucleotide changes in whole genome sequencing data&#160;for mapping20.
It was previously reported that an epifluorescent microscope is used for miniSOG-mediated cell ablation21,22. The light intensity of a standard epifluorescent compound microscope was measured to be ~0.5 mW/mm2 without a lens, which is about 4x lower than that used in the standard optogenetic mutagenesis protocol. Thus, it is highly advisable to empirically determine the efficiency when using an epi-fluorescent microscope as a light source. One advantage of the LED is the stability over thousands of hours. Previous studies have shown that miniSOG is more potent in ROS-mediated cell ablation under pulsed light22, although the precise mechanism remains to be determined. We used a digital function generator/amplifier to make pulsed light. Another option is to connect the light source to a computer using a USB-TTL interface (Figure 1A) to regulate the LED light by a program. The LED system described here can also be used for other purposes, such as optogenetic control of excitable cells using channelrhodopsin23, cell ablation using mitochondria- or cell-membrane-targeted miniSOG17,22, and Chromophore-Assisted Light Inactivation (CALI) of proteins19. Furthermore, this optogenetic mutagenesis has the potential to be applied to other organisms, such as bacteria, yeast, fly, and zebrafish, as well as mammalian cell lines.

Forward genetic screens have been widely used to generate genetic mutants disrupting various biological processes in model organisms such as Caenorhabditis elegans (C. elegans)1. Analyses of such mutants lead to the discovery of functionally important genes and their signaling pathways1,2. Traditionally, mutagenesis in C. elegans is achieved using mutagenic chemicals, radiation, or transposons2. Chemicals such as ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) are toxic to humans; gamma-ray or ultraviolet (UV) radiation mutagenesis requires special equipment; and transposon-active strains, such as mutator strains3, can cause unnecessary mutations during maintenance. We have developed a simple approach to induce heritable mutations using a genetically-encoded photosensitizer4.
Reactive Oxygen Species (ROS) can damage DNA5. The mini Singlet Oxygen Generator (miniSOG) is a green fluorescent protein of 106 amino acids that was engineered from the LOV (Light, Oxygen, and Voltage) domain of Arabidopsis phototropin 2 6. Upon exposure to blue light (~450 nm), miniSOG generates ROS including singlet oxygen with &#64258;avin mononucleotide as a cofactor6-8, which is present in all cells. We constructed a His-mSOG fusion protein by tagging miniSOG to the C-terminus of histone-72, a C. elegans variant of Histone 3. We generated a single-copy transgene9 to express His-mSOG in the germline of&#160;C. elegans. Under normal culture conditions in the dark, the His-mSOG transgenic worms have normal brood size and life span4. Upon exposure to blue LED light, the His-mSOG worms produce heritable mutations among their progeny4. The spectrum of induced mutations includes nucleotide changes, such as G:C to T:A and G:C to C:G, and small chromosome deletions4. This mutagenesis procedure is simple to perform, and requires minimal lab safety setup. Here, we describe the LED illumination setup and procedures for optogenetic mutagenesis.

<table border="0" cellpadding="0" cellspacing="0" width="626">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Ultra High Power LED light source</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHP-mic-LED-460</td>
			<td style="width:184px;">460 &#177; 5 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LED controller</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHPLCC-01</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Digital function generator/amplifier</td>
			<td style="width:71px;">PASCO</td>
			<td style="width:115px;">PI-9587C</td>
			<td style="width:184px;">PI-9587C is no longer available. The replacement is PI-8127.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">BNC cable male/male</td>
			<td style="width:71px;">THORLABS</td>
			<td style="width:115px;">CA3136</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">USB-TTL interface</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;"></td>
			<td style="width:184px;">Optional</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Photometer</td>
			<td style="width:71px;">THORLABS</td>
			<td style="width:115px;">PM50 and Model D10MM</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:115px;">1001-110</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:256px;">Copper chloride dihydrate (CuCl2&#8729;2H2O)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:115px;">C6641</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Stereomicroscope</td>
			<td style="width:71px;">Leica</td>
			<td style="width:115px;">MZ95</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="163">
			<td height="163" style="height:163px;width:256px;">NGM plate</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">Dissolve 5g NaCl, 2.5g Peptone, 20g Agar, 10 &#181;g/ml cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH6.0).&#160;</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:256px;">Lysis solution</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">10 mM Tris(pH8.8), 50 mM KCl, 0.1% Triton X100, 2.5 mM MgCl2, 100 &#181;g/ml Proteinase K</td>
		</tr>
	</tbody>
</table>

optogenetic random mutagenesis using histone-minisog in c. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

We performed a forward genetic screen with CZ20310 juSi164 unc-119(ed3) using 30 min light exposure following the standard protocol described in Sections 2 and 3. Among 60 F1 plates corresponding to 120 mutagenized haploid genomes, we found 8 F1 plates with F2 worms displaying visible phenotypes such as body size defect (Figure 3B), uncoordinated movement (Figure 3C), and adult lethality (Figure 3D). We did not notice any difference in the number of mutants between &#39;Day1&#39;&#160;and &#39;Day2&#39; plates. All progeny of singled small F2 worms and uncoordinated F2 worms showed the corresponding phenotypes, suggesting that they are heritable mutants. The full observation is summarized in Table 2. On average, a clonal screen with 120 haploid genomes will result in about 10 F1 plates containing worms with visibly detectable phenotypes such as body shape and locomotion that easily spotted under a stereomicroscope. More quantitative analysis is necessary to determine the mutation rate precisely4. The expected number of visible mutants from this screen suggests that the strain and setup are working properly for optogenetic mutagenesis.
<img alt="Figure 1" src="/files/ftp_upload/54810/54810fig1.jpg" /> 
Figure 1: The LED Setup. (A) The whole system. The LED was mounted on a custom-made holder. Details are found in the List of Materials. (B) The cover is made of plastic. It does not cover the system completely to avoid heat accumulation. (C) Controller and function generator. (D) The back side of the system. (E) An unseeded plate with CuCl2-soaked filter paper for blue light treatment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54810/54810fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54810/54810fig2.jpg" /> 
Figure 2: Experimental Procedure for a Clonal Screen. After blue light treatment, P0 worms are transferred to a seeded plate and allowed to lay eggs. P0 worms are transferred to a new seeded plate 1 d after light treatment and killed 2 d&#160;after light treatment. F1 worms are singled from the P0 plates. Phenotypes of F2 worms are examined after they become adults. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54810/54810fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54810/54810fig3.jpg" /> 
Figure 3: Example of Mutants. (A) Original strain for optogenetic mutagenesis: CZ20310 juSi164 unc-119(ed3). This strain has normal body shape, behavior, and brood size. Scale bar: 0.5 mm. (B - D) F2 progeny showing body size defect&#160;(B), uncoordinated (C), and lethal (D) phenotypes. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54810/54810fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54810/54810table1.jpg" /> 
Table 1: Genotyping of juSi164 and unc-119(ed3).
<img alt="Table 2" src="/files/ftp_upload/54810/54810table2.jpg" /> 
Table 2: Example of Phenotypes from a Clonal Screen.

Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

Optogenetyczna mutageneza losowa z użyciem Histonu-miniSOG u C. elegans | Genetyka | Wideo

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

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9.0K Views.  University of California in San Diego and Howard Hughes Medical Institute. Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Video: Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...