Name: Video: A Customizable Protocol for String Assembly gRNA Cloning STAgR
Uploaded: 2018-12-26T13:00:00.000+00:00
Duration: 10 min
Description: 9.6K Views. Ludwig Maximilian Universitat, BioMedical Center. Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

The authors would like to thank Prof. Dr. Stephan Beck and Prof. Dr. Jovica Ninkovic for their input, help and support in developing the STAgR method. Tobias Kl&#246;tzer, Anna K&#246;ferle and Valentin Baumann for helpful comments. The work has been supported by DFG (STR 1385/1-1).

1. Design of STAgR Cloning Strategy
<ol>
	<li>Decide on how many gRNA cassettes will be included in one expression vector and in which order. Determine which promoters and gRNA scaffolds should be used for each of the gRNAs. Design the gRNAs by using any online tool (e.g., www.benchling.com).</li>
</ol>
2. Design of STAgR Cloning Primers with Overhangs
NOTE: The sense protospacer sequence of the last gRNA and the antisense of the first gRNA respectively are added to the PCR primers used for amplification of the vector backbone (Figure 1). The remaining sequences are amplified from the strings, thereby attaching the sense and antisense gRNAs in the desired order. The first string piece is amplified with a forward primer containing the first gRNA N20 sequence as an overhang and a reverse primer with the second gRNA N20 sequence (reverse complement) as an overhang. All the following pieces are generated accordingly.
<ol>
	<li>Add N20 gRNA sequences to the forward amplification primers for STAgR DNA string as overhangs (Table 1). Add sense gRNA sequences 5&#39; to the forward primer &#34;scf-FP&#34; (for a conventional scaffold) or &#34;sam-FP&#34; (for a MS2 containing scaffold). FP primers anneal to the scaffold/MS2 part of the respective STAgR string (Figure 1).</li>
	<li>Add the reverse complement N20 gRNA sequence to the reverse primer sequences. Choose RP primers depending on the specific promoters and strings used (hU6-RP, mU6-RP, 7sK-RP, H1-RP).</li>
</ol>
3. Generation of STAgR Cloning Fragments
<ol>
	<li>To generate the individual cloning fragments for Gibson assembly, perform PCRs by setting up 10 &#181;L of high fidelity (HF) buffer, 1 &#181;L of 10 mM dNTPs, 0.25 &#181;L of overhang-primer (100 &#181;M), 10 ng of the DNA template (string or vector), 0.5 &#181;L HF polymerase, 1.5 &#181;L of dimethyl sulfoxide (DMSO) and add H2O to make a final volume of 50 &#181;L. 
	NOTE: Use the plasmid &#34;STAgR_Neo&#34; as a template for the backbone PCR to incorporate the gRNA scaffold to the last gRNA, if the SAM Scaffold is chosen, use &#34;STAgR_SAM&#34;. For a STAgR plasmid with six gRNA cassettes for example, six PCRs are necessary, five with DNA strings as templates and one with the vector backbone as the template.</li>
	<li>Incubate reactions on a thermocycler using the following conditions: 1 cycle of 98 &#176;C for 1 min 30 s; 38 cycles of 98 &#176;C for 10 s, 59 &#176;C (for gRNA scaffold)/ 68 &#176;C (for SAM loop) for 10 s, 72 &#176;C for 30 s (for inserts) / 1 min 30 s (for vectors); 1 cycle of 72 &#176;C for 10 min.</li>
	<li>Remove 5.5 &#181;L from the PCR reaction, add loading dye and analyze the amplified fragments on a 1% agarose gel. Load an appropriate DNA ladder for sizing (100 bp DNA ladder/1 kb DNA ladder) and run the gel in an appropriate gel running buffer (e.g., TLE buffer) at 120 V for 30 min.</li>
	<li>While the gel is running, add 5 &#181;L of the buffer provided with the restriction enzyme and 0.5 &#181;L DpnI (10 units) to the remaining 44.5 &#181;L PCR reaction and incubate for 30 min to 1 h at 37 &#176;C.</li>
	<li>Perform DNA purification using magnetic beads.
	<ol>
		<li>Add 1.8 &#181;L magnetic beads per 1 &#181;L of PCR product and mix by pipetting up and down.Incubate for 2 min at room temperature (RT).</li>
		<li>Separate beads and DNA fragments from residual liquid with a magnet. Wash beads twice with 70% ethanol by rinsing the pellet without resuspending completely.</li>
		<li>Remove all ethanol with a pipette and let the pellet air dry.</li>
		<li>Dissolve the pellet in 15&#8211;20 &#181;L H2O by pipetting up and down and separate the beads from the liquid by using a magnet. Transfer the clear supernatant to a new tube. 
		NOTE: Alternatively, column-based reaction clean-up kits can be used.</li>
	</ol>
	</li>
	<li>Determine DNA concentrations using a spectrophotometer as described elsewhere29.</li>
</ol>
4. Gibson Assembly Reaction and Bacterial Transformation
NOTE: The following steps are adapted from Gibson et al.30.
<ol>
	<li>Prepare a homemade Gibson reaction mix or use a commercial Gibson cloning kit.
	<ol>
		<li>Combine 3 mL of 1 M Tris (Tris(hydroxymethyl)aminomethane)-HCl at pH 7.5, 300 &#181;L of 1 M MgCl, 60 &#181;L of 100 mM dGTP (deoxyguanosine triphosphate), 60 &#181;L of 100 mM dATP (deoxyadenosine triphosphate), 60 &#181;L of 100 mM dTTP (deoxythymidine triphosphate), 60 &#181;L of 100 mM dCTP (deoxycytidine triphosphate), 300 &#181;L of 1 M DTT (dithiothreitol), 1.5 g of PEG-8000 (polyethylene glycol), 300 &#181;L of 100 mM NAD (nicotinamide adenine dinucleotide) and add H2O to obtain 6 mL of 5&#215; isothermal reaction buffer. 
		NOTE: Aliquoted buffer can be stored at -20 &#176;C for at least 12 months.</li>
		<li>For the Gibson assembly master mix, combine 320 &#181;L of 5x isothermal reaction buffer with 697 &#181;L of H2O and add 3 &#181;L of 10 U/&#181;L T5 exonuclease, 20 &#181;L of 2 U/&#181;L DNA polymerase and 160 &#181;L of 40 U/&#181;L Taq DNA ligase. 
		NOTE: This mix can be aliquoted and stored at -20 &#176;C for at least 12 months.</li>
	</ol>
	</li>
	<li>Use 7.5 &#181;L of assembly master mix with 2.5 &#181;L of insert and vector. For a 2x STAgR reaction use a molar vector to insert ratio of 1:1 but not more than 0.2 pmol of DNA in total. For more than 2 gRNA expression cassettes, use a vector to insert ratio of 1:3 but do not exceed a total DNA amount of 0.5 pmol. 
	NOTE: All amplified gRNA expression cassettes should be used in equimolar amounts. Include a plasmid control with the identical amount of vector but no inserts to control for undigested template vector (and/or self-ligation).</li>
	<li>Incubate samples at 50 &#176;C for 45 to 60 min. Store samples on ice or at -20 &#176;C for subsequent transformation. 
	NOTE: The protocol can be paused here.</li>
	<li>Directly transform half of the mix into chemically competent bacteria.
	<ol>
		<li>Thaw chemically competent TOP10 E. coli bacteria on ice.</li>
		<li>Add 5 &#181;L of the Gibson mix to 50 &#181;L of competent bacteria. Gently mix by flicking the bottom of the tube. Incubate on ice for 30 min.</li>
		<li>Perform a heat shock by placing the tube in a 42 &#176;C water bath or a heat block for 45 s.</li>
		<li>Put the tubes back on ice. Add 250 &#181;L of SOC medium to the bacteria and let them recover at 37 &#176;C in a shaking incubator for 30&#8211;45 min.</li>
	</ol>
	</li>
	<li>After recovery, plate the transformed bacteria onto 1.5% lysogeny broth (LB) agar plates containing ampicillin (100 &#181;g/ mL) and incubate overnight at 37 &#176;C.</li>
</ol>
5. Selecting STAgR Clones by Bacterial Colony PCR
<ol>
	<li>Prepare at least two sets of 200 &#181;L PCR reaction tubes (between 3 and 24) for each construct, which are labeled identically.</li>
	<li>Fill one set with 100 &#181;L LB medium containing 100 &#181;g/ mL ampicillin.</li>
	<li>Use a sterile pipette tip to scratch off the biological material of one bacterial colony and spread it at the bottom of the empty 100 &#181;L PCR reaction tube. Immediately transfer the tip to the second corresponding reaction tube containing the LB medium.</li>
	<li>Swirl the tip around gently to ensure some of the bacteria are transferred to the LB media and incubate at 37 &#176;C for later use.</li>
	<li>Set up a PCR master mix (10 &#181;L per reaction).
	<ol>
		<li>For 10 reactions, combine 10 &#181;L Taq buffer, 2 &#181;L 10 mM dNTPs, 0.5 &#181;L of primer (100 &#181;M), 0.5 &#181;L of Taq polymerase and add H2O to a volume of 100 &#181;L.</li>
		<li>Use the following primers: StAgR_seq_fwd2: ACTGGATCCGGTACCAAGG, StAgR_seq_rev: TTACGGTTCCTGGCCTTTTG.</li>
	</ol>
	</li>
	<li>Add 10 &#181;L of the PCR master mix to the labeled PCR reaction tubes without the LB media.</li>
	<li>Incubate reactions on a thermocycler using the following conditions: 1 cycle of 94 &#176;C for 5 min, 38 cycles of 94 &#176;C for 30 s, 55 &#176;C for 30 s, 72 &#176;C for 2 min; 1 cycle of 72 &#176;C for 10 min. 
	NOTE: Elongation time (72 &#176;C step) should be increased with the number of gRNA expression cassettes. Calculate theoretical size of inserts using Table 2 and use 1 min per 1 kb at 72 &#176;C.</li>
	<li>Add loading dye and analyze the amplified fragments on a 1% agarose gel. Load an appropriate DNA ladder for sizing (1kb DNA Ladder) and run the in appropriate gel running buffer (e.g., TLE Buffer) at 120 V for 30 min.</li>
	<li>Calculate the theoretical size of the amplicon with the help of Table 2 by adding up the individual sizes of used promoters, gRNA scaffolds and number of N20 sequences. From the results of the colony PCR, identify the bacterial clones based on the correct band size and whether they are likely to harbor correct vectors.</li>
	<li>Inoculate a 2.5 mL overnight LB culture (with 100 &#181;g/mL ampicillin) with the corresponding 100 &#181;L culture, set up earlier. Incubate at 37 &#176;C for 12 h.</li>
	<li>Extract plasmid DNA using a commercial plasmid mini kit and manufacturer&#39;s instructions31.</li>
	<li>Sequence the plasmids using the following primers: StAgR_seq_fwd1 (GAGTTAGGGGCGGGACTATG), StAgR_seq_fwd2 (ACTGGATCCGGTACCAAGG) and StAgR_seq_rev (TTACGGTTCCTGGCCTTTTG) by Sanger Sequencing.</li>
</ol>

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The authors have nothing to disclose.

Here we present a detailed protocol for STAgR allowing the fast and simple generation of gRNA expression plasmids of varying sizes. This protocol can be used for one-step generation of gRNA plasmids with 2&#8211;8 gRNA expression cassettes (with or without two MS2 RNA aptamers) into the gRNA expression vector STAgR_Neo, and the use of human U6, mouse U6, human 7sK and human H1 promoters28,32,33. If more than four cassettes are desired, it is recommended to use at least two different promoter/scaffold types to increase efficiency. Other vectors, promoters and promoter combinations as well as more than 8 gRNA cassettes might be feasible as well; however, this has not been tested. Although in our experience the method works reliably and reproducibly, we have determined critical steps and troubleshooting strategies should the expected outcome not immediately occur. In our experience some steps are pivotal for the success of the approach: first and foremost, it is important to use the correct molar ratios of inserts to the vector backbone at the Gibson assembly step (3:1). However, we noticed that for some gRNA sets a 1:1 ratio of inserts to vector is beneficial, even when the gRNA cassette number does exceed two. Secondly, the period of the Gibson assembly reaction should be sufficiently long (at least 45 min recommended).
STAgR can be employed with a large number of modifications. Targeting sequences, gRNA numbers and scaffolds, promoters and vector backbones can be customized and freely combined. However, each combination may have slightly different requirements. In our experience, if medium or high numbers of gRNA cassettes (&#62;4) are desired but not obtained immediately, usually the fastest way to overcome this issue is to change the order of the individual gRNA cassettes in the vector. Similarly, combining different gRNA promoters and scaffolds improves efficiency (and thus lowers the required number of colony PCRs that have to be scored) when many gRNA cassettes are cloned. We found that incorrect clones are usually generated by the spontaneous conjunction of repeated sequences during Gibson assembly. Although this can reduce the efficiency of the method, this also results in the simultaneous generation of vectors with functional gRNA subsets28.
Generation of multiplexing gRNA vectors containing more than one gRNA expression cassette has been reported with a number of different methods21,22,23,24,25,26,27. While some employ simultaneous or sequential cloning of gRNA pairs22,34, others depend on Golden Gate assembly and several sequential cloning steps25,35. A special approach has been used by Xie et al., by employing the endogenous cellular tRNA processing system to cut several gRNAs out of a single progenitor transcript36. The advangetage of this method is the requirement of only one promoter; the drawback is the need of newly synthesized DNA fragments for each gRNA combination. Each gRNA multiplexing method has advantages and disadvantages; STAgR combines simplicity with efficiency, high number of possible gRNA cassettes with low cost.
STAgR (and other multiplexing systems) will likely be instrumental in enabling efficient (and simultaneous) disruption of multiple genes in vivo, as pioneered in zebrafish brains28. Another future application of gRNA multiplexing vectors is the promise for manipulation of complete transcriptional programs in contrast to the induction or repression of single genes. Likely, these approaches will allow transforming cells and organs at will to a much higher degree than it is possible today.

String assembly gRNA cloning (STAgR) is a method enabling efficient overnight generation of expression vectors containing multiple gRNA expression cassettes in one single cloning step. The STAgR protocol does not depend on specialist expertise or materials and offers multiple options for customization.
The bacterial CRISPR protein Cas9 has a unique capacity. It is able to find and selectively bind only those DNA sequences encoded in naturally occurring crRNAs or engineered gRNAs1. Its utilization as a molecular tool has enabled a large number of approaches that were impossible, unfeasible or only limited to certain cellular models until recently. These include targeted gene mutations2, genome engineering3, epigenome editing4,5,6, transcriptional activation and gene silencing7,8. As far as we know CRISPR is universally deployable, as reports of its application exist for a wide range of target sites, cell types and species. However, many CRISPR applications depend directly or indirectly on the delivery of multiple gRNAs including a series of genomic manipulations (translocations9,10, medium11 and large scale genomic alterations12,13), genome engineering (use of Cas9 nickases14, generation of conditional alleles11,15 or serial mutations16) and tracing strategies17. Moreover, for other approaches (transcriptional engineering18, epigenome engineering19,20) multiple targeting sites are not strictly mandatory, however they reach their maximum effect often only under this condition.
Several useful methods have been described to generate gRNA vectors containing more than one gRNA expression cassette21,22,23,24,25,26,27. Here, we present a protocol for STAgR, string assembly gRNA cloning, which is outstanding in its combination of simplicity and speed (only one overnight cloning step is needed), low cost (as DNA templates can be reused multiple times), customizability (for different gRNA systems and promoters) and effectiveness (it allows the generation of vectors containing high numbers of gRNA cassettes)28.
STAgR can in principle be used to generate expression vectors with few (1&#8211;2 gRNAs), several (3&#8211;5 gRNAs) and some of the highest number of expression cassettes reported so far (6&#8211;8 gRNAs)28. Similarly, STAgR is applicable for any gRNA sequence, backbone or promoter. Since however, STAgR is mainly based on polymerase chain reactions (PCR) and Gibson assembly, gRNAs with high sequence similarities should be generated using an alternative method.

<table border="0" cellpadding="0" cellspacing="0" style="width:929px;" width="929">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Phusion High- Fidelity DNA Polymerase</td>
			<td style="width:139px;">NEB</td>
			<td style="width:119px;">M0530L</td>
			<td style="width:403px;">Polymerase for Gibson Master Mix as well as all PCRs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">dNTPs</td>
			<td style="width:139px;">Life Technology</td>
			<td style="width:119px;">R0191</td>
			<td>dNTPs for all PCRs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">dATP</td>
			<td style="width:139px;">Life Technology</td>
			<td style="width:119px;">R0141</td>
			<td>dNTPs for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">dCTP</td>
			<td style="width:139px;">Life Technology</td>
			<td style="width:119px;">R0151</td>
			<td>dNTPs for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">dGTP</td>
			<td style="width:139px;">Life Technology</td>
			<td style="width:119px;">R0161</td>
			<td>dNTPs for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">dTTP</td>
			<td style="width:139px;">Life Technology</td>
			<td style="width:119px;">R0171</td>
			<td>dNTPs for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Agarose</td>
			<td style="width:139px;">VWR</td>
			<td style="width:119px;">443666A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DpnI</td>
			<td style="width:139px;">NEB</td>
			<td style="width:119px;">R0176S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">AMPure XP Beads</td>
			<td style="width:139px;">Beckman Coulter</td>
			<td style="width:119px;">A63881</td>
			<td>for DNA clean up</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tris HCL</td>
			<td style="width:139px;">Roth</td>
			<td style="width:119px;">9090.2</td>
			<td>for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DTT</td>
			<td style="width:139px;">Life Technology</td>
			<td style="width:119px;">R0861</td>
			<td>for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PEG 8000</td>
			<td style="width:139px;">VWR</td>
			<td style="width:119px;">RC- 077</td>
			<td>for Gibson Master Mix</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">NAD</td>
			<td style="width:139px;">Cayman Chemicals Company</td>
			<td align="right" style="width:119px;">16077</td>
			<td>for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">T5 Exonuclease</td>
			<td style="width:139px;">NEB</td>
			<td style="width:119px;">M0363S</td>
			<td>for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Taq DNA Ligase</td>
			<td style="width:139px;">NEB</td>
			<td style="width:119px;">M0208S</td>
			<td>for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Ampicilin</td>
			<td style="width:139px;">SigmaAldrich</td>
			<td style="width:119px;">PHR1393-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DNA Ladder 1kb Plus</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SM0311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Plasmid Mini Kit</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">K0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Plasmid and Primer Sequences</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>All sequences available at Figshare (https://goo.gl/UGdc3Q).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">EDTA</td>
			<td style="width:139px;">PanReacAppliChem</td>
			<td style="width:119px;">6381-92-6</td>
			<td>for TLE Buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">MgCl2</td>
			<td style="width:139px;">SigmaAldrich</td>
			<td style="width:119px;">M8266</td>
			<td>for Gibson Master Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Top10 chemically competent bacteria</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">C404003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">S.O.C. Medium</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">15544034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Yeast- Extract</td>
			<td style="width:139px;">SigmaAldrich</td>
			<td style="width:119px;">Y1625</td>
			<td>for lysogenic broth (LB Media)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">NaCl</td>
			<td style="width:139px;">SigmaAldrich</td>
			<td style="width:119px;">S7653</td>
			<td>for lysogenic broth (LB Media)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Peptone</td>
			<td style="width:139px;">SigmaAldrich</td>
			<td align="right" style="width:119px;">91249</td>
			<td>for lysogenic broth (LB Media)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Agar-Agar</td>
			<td style="width:139px;">SigmaAldrich</td>
			<td align="right" style="width:119px;">5040</td>
			<td>for lysogenic broth (LB Media)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Taq DNA Polymerase</td>
			<td style="width:139px;">Quiagen</td>
			<td align="right" style="width:119px;">201203</td>
			<td></td>
		</tr>
	</tbody>
</table>

a customizable protocol for string assembly grna cloning (stagr)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

Following the protocol at hand makes the generation of customized multiplexing gRNA vectors feasible (Figure 1). Representative results of STAgR approaches using six different gRNA cassettes are depicted in Figure 2. Figure 2A shows a typical outcome of the PCRs used to obtain the STAgR pieces (protocol 3.1-3.3). The six amplicons (BB, S1-S5) represent linear DNA pieces containing the individual gRNA N20 sequences on their ends. The plasmid backbone (BB) is now extended with the targeting sequences of gRNA1 and gRNA6 and therefore possesses the required overlaps to two other PCRs (S1 and S5) for Gibson assembly (Figure 2A, Table 3). After purification a DNA yield of at least 1 &#181;g for vectors and inserts can be achieved.
After Gibson assembly, bacterial transformation should result in 100 to 700 bacterial colonies. Figure 2B shows a representative analysis of 10 bacterial colonies via colony PCR, following a STAgR protocol with six gRNA cassettes. Gel electrophoresis indicates that three clones show the expected amplicon size for full assembly (Table 2). Other clones likely received STAgR vectors containing one to five gRNA cassettes, whereas one clone (Figure 2B, No.18) is empty.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58556/58556fig1.jpg" /> 
Figure 1: Overview of STAgR primer design. First gRNA number and scaffolds are chosen, then gRNA order and promoter and last, which type of vector should be used. For each gRNA, a specific forward primer with the gRNA targeting sequence in sense orientation and either the common part of &#34;scf-FP&#34; or &#34;sam-FP&#34; is designed. Similarly, to generate the individual reverse primer the corresponding promoter sequence (RP) is fused with the reverse complement of the N20 sequence of the next gRNA. <a href="https://www.jove.com/files/ftp_upload/58556/58556fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58556/58556fig2.jpg" /> 
Figure 2: Representative results of STAgR approaches using six different gRNA cassettes. (A) Representative example of a PCR of five inserts as well as the vector STAgR_Tomato. (B) Colony PCR of a 6x STAgR reaction using hU6 and mU6 promoters and canonical gRNA as well as SAM scaffolds. 22 bacterial colonies were analyzed, of which 7 showed the expected band indicating complete assembly. Ladder: 1 kb plus, numbers in base pairs (bp). <a href="https://www.jove.com/files/ftp_upload/58556/58556fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Forward primer for String and Vector</td>
			<td></td>
		</tr>
		<tr>
			<td>scaffold_fwd</td>
			<td>GTTTTAGAGCTAGAAATAGCAAGTT</td>
		</tr>
		<tr>
			<td>SAM_fwd</td>
			<td>GTTTTAGAGCTAGGCCAACATGAGG</td>
		</tr>
		<tr>
			<td>Reverse primer for String and Vector</td>
			<td></td>
		</tr>
		<tr>
			<td>hU6_rev</td>
			<td>CGGTGTTTCGTCCTTT</td>
		</tr>
		<tr>
			<td>mU6_rev</td>
			<td>CAAACAAGGCTTTTCTCCAAGG</td>
		</tr>
		<tr>
			<td>hH1_rev</td>
			<td>CTGGGAAAGAGTGGTCTCATACAGA</td>
		</tr>
		<tr>
			<td>h7SK_rev</td>
			<td>CCGAGGTACCCAAGCGG</td>
		</tr>
	</tbody>
</table>
Table 1: Primer sequences for gRNA scaffolds and promoters.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Sizes of promoters and scaffolds.</td>
			<td></td>
		</tr>
		<tr>
			<td>hU6</td>
			<td>265 bp</td>
		</tr>
		<tr>
			<td>hH1</td>
			<td>225 bp</td>
		</tr>
		<tr>
			<td>mU6</td>
			<td>316 bp</td>
		</tr>
		<tr>
			<td>h7SK</td>
			<td>245 bp</td>
		</tr>
		<tr>
			<td>gRNA scaffold</td>
			<td>83 bp</td>
		</tr>
		<tr>
			<td>SAMloop + gRNA scaffold</td>
			<td>143 bp</td>
		</tr>
	</tbody>
</table>
Table 2: Theoretical sizes of each expression cassette to calculate colony PCR outcome. 
<table>
	<tbody>
		<tr>
			<td></td>
			<td>hU6</td>
			<td>hH1</td>
			<td>mU6</td>
			<td>h7SK</td>
			<td>STAgR_Neo</td>
			<td>STAgR_tdTomato</td>
		</tr>
		<tr>
			<td>gRNA Scf</td>
			<td>400 bp</td>
			<td>360 bp</td>
			<td>451 bp</td>
			<td>380 bp</td>
			<td>3722 bp</td>
			<td>4487 bp</td>
		</tr>
		<tr>
			<td>SAM</td>
			<td>460 bp</td>
			<td>420 bp</td>
			<td>511 bp</td>
			<td>440 bp</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 3: Sizes of promoters and scaffolds.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>hU6</td>
			<td>hH1</td>
			<td>mU6</td>
			<td>h7SK</td>
			<td>STAgR_Neo</td>
			<td>STAgR_tdTomato</td>
		</tr>
		<tr>
			<td>gRNA Scf</td>
			<td>368 bp</td>
			<td>328 bp</td>
			<td>419 bp</td>
			<td>348 bp</td>
			<td>+74 bp</td>
			<td>+67 bp</td>
		</tr>
		<tr>
			<td>SAM</td>
			<td>428 bp</td>
			<td>388 bp</td>
			<td>479 bp</td>
			<td>408 bp</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 4: Calculated sizes of STAgR pieces after PCR.

Watch this Scientific Journal Video about A Customizable Protocol for String Assembly gRNA Cloning STAgR at JoVE.com

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

a-customizable-protocol-for-string-assembly-grna-cloning-stagr

Nanyang Technological University

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JoVE Journal

Genetics

9.6K Views.  Ludwig Maximilian Universitat, BioMedical Center. Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

Video: A Customizable Protocol for String Assembly gRNA Cloning STAgR

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 ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...