This research was supported by National Science Foundation grant IOS-1025642 (GBM). We thank Maria Harrison for providing the ARqua1 strain.

1. Guide RNA Design and Vector Construction
<ol>
	<li>Identify target sequences for the genes of interest. There are a variety of online CRISPR target-finding programs suitable for this step13,14. 
	NOTE: Here we use the GN20GG target motif, but other designs may be suitable depending on the application or vector system used.</li>
	<li>Design 60-mer gRNA oligos to include the GN19 portion of the target motifs flanked by 5&#39; and 3&#39; 20-nt sequences that are required for DNA assembly. The final 60-mer motif is: 5&#39;-TCAAGCGAACCAGTAGGCTT-GN19-GTTTTAGAGCTAGAAATAGC-3&#39;. 
	NOTE: Standard, desalted primers work fine.</li>
	<li>Prepare the four DNAs for assembly (Figure 1). See supplementary file for DNA sequences.
	<ol>
		<li>Digest 1 - 5 &#181;g of p201N:Cas910 plasmid with the restriction enzyme SpeI in 1x Buffer 4 at 37 &#176;C for 2 hr. Column-purify the digest according to manufacturer&#39;s instructions. Resuspend in ~15 &#181;l of 10 mM Tris-HCl, and quantify by UV spectrophotometry.</li>
		<li>Perform a second digest with the restriction enzyme SwaI in 1x Buffer 3.1 at 25 &#176;C for 2 hr. Check 100 - 200 ng on a 0.8% agarose gel to confirm complete digestion. 
		NOTE: A correctly digested plasmid will have a single band at 14,313 bp. Note: Heat inactivation of the enzyme and dephosphorylation are not required. Digested plasmid may be stored at -20 &#176;C.</li>
		<li>PCR amplify the Medicago truncatula (Mt) U6 promoter and scaffold DNAs from the pUC gRNA Shuttle10 plasmid using the primers SwaI_MtU6F / MtU6R and ScaffoldF / SpeI_ScaffoldR, respectively. Use a high-fidelity polymerase with the PCR conditions: 95 &#176;C for 3 min; 31 cycles (98 &#176;C for 20 sec, 60 &#176;C for 15 sec, 72 &#176;C for 30 sec); and 72 &#176;C for 5 min.
		<ol>
			<li>Visualize ~3 &#181;l aliquots of PCR products on a 1% agarose gel to confirm amplification. The MtU6 amplicon is 377 bp and the scaffold amplicon is 106 bp. Column-purify the remaining PCR products according to manufacturer&#39;s instructions, quantify by UV spectrophotometry and store at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Resuspend the entire tube of gRNA oligos to 100 &#181;M in laboratory-grade water. Add 1 &#181;l of 100 &#181;M oligo to 500 &#181;l of 1x Buffer 2.1. Mix well. If pooling multiple oligos, add 1 &#181;l of each 100 &#181;M oligo to the 1x Buffer 2.1 tube. Diluted oligos can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Program a thermal cycler to hold at 50 &#176;C, using a heated lid. Combine 100 ng (0.011 pmol) of linearized vector, 50 ng (0.2 pmol) of MtU6 promoter, 12 ng (0.2 pmol) of scaffold, 1 &#181;l (0.2 pmol) of diluted gRNA oligo(s), and water to a volume of 5 &#181;l. Add 5 &#181;l of 2x high-fidelity DNA assembly master mix, mix well and spin down. Incubate reaction for 60 min in the 50 &#176;C thermal cycler. Then place the reaction on ice. 
	Note: Reaction volumes can be increased to accommodate low DNA yields.</li>
	<li>Transform 2 &#181;l&#160;of the reaction into competent E. coli cells using standard techniques and plate on LB supplemented with 50 mg L-1 kanamycin (Kan50). Grow plated-cells at 37 &#176;C overnight. Save the remaining DNA assembly reaction at -20 &#176;C.</li>
	<li>Colony screen by PCR using the primers StUbi3P218R and ISceIR. Dilute an aliquot of the remaining DNA assembly reaction with water 1:5. Use 1 &#181;l&#160;as a positive control for the colony screen and 1 ng of circular p201N:Cas9 plasmid as a no-insert control.
	<ol>
		<li>PCR amplify with the conditions; 95 &#176;C for 3 min; 31 cycles (95 &#176;C for 30 sec, 58 &#176;C for 30 sec, 72 &#176;C for 30 sec); and at 72 &#176;C for 5 min. Visualize PCR products on a 1% agarose gel. Ensure that correct insertions have a band at 725 bp and vectors without inserts (e.g., uncut vector) will have a 310-bp band.</li>
	</ol>
	</li>
	<li>Grow positive colonies in LB Kan50 liquid cultures overnight at 37 &#176;C and purify plasmids according to manufacturer&#39;s instructions. Sanger sequence plasmids with the StUbi3P218R primer and align chromatograms to the MtU6 promoter, target, and scaffold sequences to ensure no errors were introduced during cloning.</li>
	<li>Perform a diagnostic digest by digesting ~1 &#181;g of plasmid with EcoRV and StyI. Visualize on a 0.8% agarose gel. Fragments (in bp) are: 4192, 2001, 1743, 1544, 1381, 995, 831, 702, 460, 423, 318, and 174.</li>
	<li>Use DNA assembly to construct vectors with multiple gRNAs.
	<ol>
		<li>To construct a vector with two gRNA cassettes, PCR amplify the first-position gRNA with the primers SwaI_MtU6F / UNS1_ScaffoldR and the second-position gRNA with the primers UNS1_MtU6F / SpeI_ScaffoldR from 1 ng of each of the respective plasmids constructed in steps 1.1 - 1.8. Use the PCR conditions in step 1.3.3. Visualize ~3 &#181;l aliquots of PCR products on a 1% agarose gel to confirm amplification. Amplicons are ~500 bp.</li>
		<li>Combine and mix approximately equal amounts of un-purified PCR products in a 200 &#181;l tube (typically 3 - 4 &#181;l of each). For assembly, add 1 &#181;l combined PCR products, 50 ng of SwaI and SpeI linearized p201N:Cas9, laboratory-grade water to 2.5 &#181;l and 2.5 &#181;l of 2x high-fidelity DNA assembly master mix. Incubate in a 50 &#176;C thermal cycler for 15 min. 
		Note: The DNA assembly manuals recommends a 15 min incubation for 2 - 3 fragments.</li>
		<li>Transform 1 - 2 &#181;l into competent E. coli cells and plate on LB Kan50. Grow plated cells at 37 &#176;C overnight. Save the remaining reaction at -20 &#176;C.</li>
		<li>Colony screen by PCR with primers StUbi3P218R and ISceIR with the same conditions as step 1.6. Correct clones will have a 1,200 bp amplicon. Grow positive colonies in LB Kan50 liquid cultures overnight and purify plasmids.</li>
		<li>Sanger sequence plasmids with the primers StUbi3P218R (covers first-position gRNA) and p201R (covers second-position gRNA). Align chromatograms to the MtU6 promoter, target, and scaffold sequences. Diagnostic digest with EcoRV and StyI will result in the following fragments (in bp): 4192, 2476, 1743, 1544, 1381, 995, 831, 702, 460, 423, 318, 174. Bolded fragment contains the second gRNA.</li>
	</ol>
	</li>
	<li>After meeting all the quality-control expectations, transform plasmids into Agrobacterium rhizogenes strain ARqua115. Add 1 &#181;l of the plasmid prep to 50 &#181;l of electrocompetent cells and electroporate in a 1 mm cuvette with the settings: 2.4 kV, 25 &#181;F, and 200 &#8486;. Recover cells in ~500 &#181;l of SOC and shake at 28 &#176;C for 2 hr. Plate on LB Kan50 and grow at 28 &#176;C for 2 days. Perform colony screen using the same primers and PCR conditions as 1.3.3. Make glycerol stocks from positive clones.</li>
</ol>
2. Hairy Root Transformation
<ol>
	<li>Sterilize tomato seeds in 20% household bleach for 15 min, with constant mixing. In a laminar flow hood, remove the bleach and wash in sterile laboratory-grade water 3 times.</li>
	<li>Plate 30 seeds in a GA-7 box containing &#189; MS media (2.22 g L-1 MS salts + Gamborg Vitamins, 10 g L-1 sucrose, 3 g L-1 gellan gum, pH 5.8). Germinate for ~2 days in the dark, then move GA-7 boxes to light. Grow seedlings for ~4 more days in the light.</li>
	<li>The day before transformation, streak out A. rhizogenes cultures on solid LB Kan50 and grow at 28 &#176;C overnight.</li>
	<li>Day of transformation, in the laminar flow hood assemble sterile materials: 12 ml culture tubes, 50 ml conical tube, &#189; MS liquid (no gellan gum), 200 &#181;M acetosyringone, filter paper, Petri dishes, forceps and scalpel, pipettes and pipette tips. Add 25 &#181;l of acetosyringone to 50 ml of &#189; MS and pour ~ 6 ml into each culture tube.</li>
	<li>Use a bent 200 &#181;l tip to scrape A. rhizogenes cells from the plate and resuspend in the 6 ml of &#189; MS liquid. Vortex the tube to completely resuspend the cells. After resuspending cells from each vector, measure the optical density (O.D.) of 1 ml of cells at a fixed wavelength of 600 nM. Ensure O.D. is between 0.2 and 0.4; dilute or add more cells as necessary. Repeat for each construct.</li>
	<li>Add ~ 2 ml of &#189; MS liquid to a Petri dish with a sterile filter paper. Excise cotyledons from the seedlings and place on the moistened filter paper. Once all cotyledons have been collected, cut the distal ~1 cm off of the cotyledons, resulting in cotyledon pieces with two cut ends. Add explants to A. rhizogenes solutions, mix, and incubate for 20 min with occasional inverting. 
	Note: Approximately 12 explants per construct are routinely used, though as many as 80 explants have been inoculated in 6 ml of A. rhizogenes solution.</li>
	<li>During the A. rhizogenes incubation, add filter paper to Petri dishes, one per construct transformed. Also set &#189; MS solid media, no antibiotics, in the laminar flow hood to dry.</li>
	<li>Scoop cotyledons out of A. rhizogenes solution with sterile forceps and place on dry filter paper. Cover with Petri lid to ensure tissues do not dry out while proceeding to the next transformation. Blot cotyledons on the filter paper and transfer, abaxial side up, to &#189; MS media. Wrap plates with surgical tape and co-cultivate in the dark at room temperature for 2 days.</li>
	<li>After co-cultivation, transfer cotyledons, abaxial side up, to &#189; MS media supplemented with 300 mg L-1 ticarcillin/clavulanic acid (Tim300, to prevent the growth of residual A. rhizogenes) and Kan50 (for plant selection) and wrap with surgical tape. Maintain cultures under fluorescent lights at room temperature with a 16-hour photoperiod.</li>
	<li>After ~1.5 - 2 weeks roots at least 2 cm in length are excised from the cotyledons using sterile forceps and a scalpel and transferred to &#189; MS Kan50 Tim300 media. Transfer 10 to 15 roots to a single plate. Mark the position of the root tips with a marker, wrap with surgical tape, and maintain in culture room.</li>
	<li>After one week, transformed roots are seen growing on the selective media. Harvest a subsample of transformed roots for DNA extraction using the preferred DNA-extraction method. Note: Plates with cotyledons can be kept for 2 - 3 weekly harvests, after which point the roots grow into a tangled mess and are difficult to isolate. Non harvested root tissues can be maintained indefinitely. A variety of assays can be performed on the isolated DNA samples to determine DNA mutation frequency and type. Results from a few such assays are described below.</li>
</ol>

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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+a+CRISPR/Cas9+library+for+functional+genomics+in+human+cells.">High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.</a> Nature. 509 (509), 487-491 (2014).</li><li>Doudna, J. A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346 (6213), (2014).</li><li>Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Patron, N. 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The authors have nothing to disclose.

Since DNA assembly is used to recombine any overlapping DNA sequences, this cloning method can be applied to any CRISPR vector construction. Most CRISPR cloning schemes use either gene synthesis of the gRNA, type IIS restriction enzymes17,18, or overlap-extension PCR19. Each of these techniques has inherent advantages and disadvantages, but they typically require multiple hands-on cloning steps. The primary advantage of the cloning method presented here is that the entire process occurs in a single,...

The ability to generate targeted DNA modifications with CRISPR/Cas9 has great potential for functional genomics studies. There are two components of the CRISPR/Cas9 system; the Cas9 nuclease, derived from Staphylococcus pyogenes and an approximately 100-nt guide RNA (gRNA) molecule that directs Cas9 to the targeted DNA site(s)1. Target recognition is conferred by the first ~20-nt of the gRNA, which allows for high-throughput production of targeting vectors2,3. Most organisms that can be engineered, already have been with CRISPR/Cas9 technology4,5.
In plants, constitutive promoters, such as the CaMV 35S promoter, are commonly used to drive the expression of the Cas9 nuclease6. The gRNAs are expressed using the RNA polymerase III U6 or U3 promoters which restricts the first base of the gRNA to either a G, for U6, or A for U3, for efficient transcription. However RNA polymerase II promoters, which are free of these restrictions, have also been used7,8.
Different gRNAs induce DNA mutations with different efficiencies, and so it can be important to first validate CRISPR vectors before investing in whole-plant transformations or setting up extensive phenotypic screens. Transient expression of CRISPR constructs in plants, by using agroinfiltration for example, generally results in a lower frequency of DNA modification as compared to stable plants6, making detection of mutations difficult and phenotypic assays impractical with such approaches. So-called hairy roots are a convenient, alternative system since a large number of independent, stably transformed materials can be generated within weeks, as opposed to months for stable plants. CRISPR vectors are very effective at inducing DNA mutations in hairy roots9,10.
DNA assembly methods efficiently ligate DNA fragments containing overlapping ends11. A major advantage of some DNA assembly methods is the ability to incorporate ssDNA (i.e., oligos) into the assembled products. Since gRNAs are only ~20-nt long and new targets can be made with synthesized oligos, these DNA assembly methods are well suited to CRISPR cloning. The protocols described here are based on the p201 series of CRISPR vectors that has been successfully used in soybean10, poplar12 and now tomato. The cloning procedure presented offers several advantages over the current cloning method10. Namely, fully functional vectors can be generated in a single cloning reaction in a single day. Vector construction can also be pooled to generate multiple CRISPR vectors in parallel, further reducing hands-on time and material costs. We also present a protocol for the generation of tomato hairy roots as an efficient method to produce transgenic materials with targeted gene deletions. Hairy roots are used to validate the CRISPR vectors and provide material for subsequent experiments.

<table border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">NEBuilder&#174; (HiFi DNA assembly mix)</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:247px;">E5520</td>
			<td style="width:157px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">p201N:Cas9</td>
			<td>Addgene</td>
			<td>59175</td>
			<td>The p201H:Cas9 plasmid (59176) is also compatible with the reported overlaps and enzymes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC gRNA Shuttle</td>
			<td>Addgene</td>
			<td>47024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SwaI</td>
			<td>New England Biolabs</td>
			<td>R0604S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SpeI</td>
			<td>New England Biolabs</td>
			<td>R0133S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymo clean and concentrator-5 column purification</td>
			<td>Zymo Research</td>
			<td>D4003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NEB Buffer 2.1</td>
			<td>New England Biolabs</td>
			<td>B7202S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NEB CutSmart (Buffer 4)</td>
			<td>New England Biolabs</td>
			<td>B7204S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NEB Buffer 3.1</td>
			<td>New England Biolabs</td>
			<td>B7203S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EconoSpin Mini Spin Column (plasmid prep)</td>
			<td>Epoch Life Sciences</td>
			<td>1910-050/250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRV-HF&#174;</td>
			<td>New England Biolabs</td>
			<td>R3195S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">StyI-HF&#174;</td>
			<td>New England Biolabs</td>
			<td>R3500S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MS Salts + Gamborg Vitamins</td>
			<td>Phytotechnology Laboratories</td>
			<td>M404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phytagel&#8482; (gellan gum)</td>
			<td>Sigma Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GA-7 Boxes</td>
			<td>Sigma Aldrich</td>
			<td>V8505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micropore&#8482; surgical tape</td>
			<td>3M</td>
			<td>1535-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Timentin&#174; (Ticarcillin/Clavulanic acid)</td>
			<td>Various</td>
			<td>0029-6571-26</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primers 5&#39; -&#62; 3&#39;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SwaI_MtU6F&#160;</td>
			<td colspan="3">GATATTAATCTCTTCGATGAAATTTATGCCTATCTTATATGATCAATGAGG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MtU6R&#160;&#160;</td>
			<td colspan="2">AAGCCTACTGGTTCGCTTGAAG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ScaffoldF&#160;</td>
			<td colspan="2">GTTTTAGAGCTAGAAATAGCAAGTT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpeI_Scaffold R</td>
			<td colspan="3">GTCATGAATTGTAATACGACTCAAAAAAAAGCACCGACTCGGTG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StUbi3P218R&#160;</td>
			<td colspan="2">ACATGCACCTAATTTCACTAGATGT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISceIR</td>
			<td colspan="2">GTGATCGATTACCCTGTTATCCCTAG</td>
			<td>Cannot be used for Sanger sequencing since there is a second binding site on the plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UNS1_Scaffold R&#160;</td>
			<td colspan="2">GAGAATGGATGCGAGTAATGAAAAAAAGCACCGACTCGGTG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UNS1_MtU6 F&#160;&#160;</td>
			<td colspan="3">CATTACTCGCATCCATTCTCATGCCTATCTTATATGATCAATGAGG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p201R&#160;</td>
			<td colspan="2">CGCGCCGAATTCTAGTGATCG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Bolded sequences denote 20-nt overlaps with linearized p201N:Cas9.</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput crispr vector construction and characterization of dna modifications by generation of tomato hairy roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

CRISPR vector construction with DNA assembly typically generates tens to hundreds of independent clones. Colony screening by PCR easily identifies correct clones and can distinguish between plasmids with and without inserts (Figure 2A) which is useful for troubleshooting. Typically, all of the clones contain an insert and a user may opt to skip the colony screening steps altogether. Diagnostic digests (Figure 2B) and Sanger sequencing are used for quality...

Watch this Scientific Journal Video about High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots at JoVE.com

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

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17.8K Views.  Boyce Thompson Institute for Plant Research. Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Video: High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...