Name: parallel high throughput single molecule kinetic assay for site-specific dna cleavage
Uploaded: 2020-05-06T15:00:00
Description: Watch this Scientific Journal Video about Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage at JoVE.com

This work was supported by the National Science Foundation grant MCB-1715317.

1. Making the flow cell
<ol>
	<li>Washing the coverslips
	<ol>
		<li>Place coverslips in staining jars and sonicate with ethanol (EtOH), then with 1 M KOH (for 30 min each). To avoid KOH precipitation in EtOH, rinse thoroughly with ddH2O between washes.</li>
		<li>Repeat both EtOH and the KOH washing steps 1x for a total number of four washes (two EtOH and two KOH). Store cleaned coverslips in ddH2O in staining jars.</li>
	</ol>
	</li>
	<li>Cut loading and exit tubes (8 cm long) using a clean razo...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn7:+a+target+site-specific+transposon.">Tn7: a target site-specific transposon.</a> Molecular Microbiology. 5 (11), 2569-2573 (1991).</li><li>Gori, J. L., 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=Delivery+and+Specificity+of+CRISPR/Cas9+Genome+Editing+Technologies+for+Human+Gene+Therapy.">Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy.</a> Human Gene Therapy. 26 (7), 443-451 (2015).</li><li>Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., Gregory, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+with+engineered+zinc+finger+nucleases.">Genome editing with engineered zinc finger nucleases.</a> Nature Reviews Genetics. 11 (9), 636-646 (2010).</li><li>Joung, J. K., Sander, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALENs:+a+widely+applicable+technology+for+targeted+genome+editing.">TALENs: a widely applicable technology for targeted genome editing.</a> Nature Reviews Molecular Cell Biology. 14 (1), 49-55 (2013).</li><li>Alves, J., Urbanke, C., Fliess, A., Maass, G., Pingoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+stopped-flow+kinetics+of+the+cleavage+of+synthetic+oligodeoxynucleotides+by+the+EcoRI+restriction+endonuclease.">Fluorescence stopped-flow kinetics of the cleavage of synthetic oligodeoxynucleotides by the EcoRI restriction endonuclease.</a> Biochemistry. 28 (19), 7879-7888 (1989).</li><li>Deng, J., Jin, Y., Chen, G., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+fluorescent+assay+for+real-time+monitoring+site-specific+DNA+cleavage+by+EcoRI+endonuclease.">Label-free fluorescent assay for real-time monitoring site-specific DNA cleavage by EcoRI endonuclease.</a> Analyst. 137 (7), 1713-1717 (2012).</li><li>Becker, W. R., 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+analysis+reveals+rules+for+target+RNA+binding+and+cleavage+by+AGO2.">High-throughput analysis reveals rules for target RNA binding and cleavage by AGO2.</a> Molecular Cell. 75 (4), 741-755 (2019).</li><li>vanden Broek, B., Lomholt, M. A., Kalisch, S. M., Metzler, R., Wuite, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+DNA+coiling+enhances+target+localization+by+proteins.">How DNA coiling enhances target localization by proteins.</a> Proceedings of the National Academy of Sciences. 105 (41), 15738-15742 (2008).</li><li>Gambino, S., 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=A+single+molecule+assay+for+measuring+site-specific+DNA+cleavage.">A single molecule assay for measuring site-specific DNA cleavage.</a> Analytical Biochemistry. 495, 3-5 (2016).</li><li>Jones, N. D., 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=Retroviral+intasomes+search+for+a+target+DNA+by+1D+diffusion+which+rarely+results+in+integration.">Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.</a> Nature Communications. 7, 11409 (2016).</li><li>van Aelst, K., 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=Type+III+restriction+enzymes+cleave+DNA+by+long-range+interaction+between+sites+in+both+head-to-head+and+tail-to-tail+inverted+repeat.">Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat.</a> Proceedings of the National Academy of Sciences. 107 (20), 9123-9128 (2010).</li><li>Williams, K., 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=A+single+molecule+DNA+flow+stretching+microscope+for+undergraduates.">A single molecule DNA flow stretching microscope for undergraduates.</a> American Journal of Physics. 79 (11), 1112-1120 (2011).</li><li>Song, D., 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=Tethered+particle+motion+with+single+DNA+molecules.">Tethered particle motion with single DNA molecules.</a> American Journal of Physics. 83 (5), 418-426 (2015).</li><li>Etson, C. M., Todorov, P., Walt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Restriction+Endonucleases+Reaction+Mechanisms+via+Dwell-Time+Distribution+Analysis.">Elucidating Restriction Endonucleases Reaction Mechanisms via Dwell-Time Distribution Analysis.</a> Biophys Journal. 106 (2), 22 (2014).</li><li>Piatt, S., Price, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+dwell+times+with+the+Generalized+Method+of+Moments.">Analyzing dwell times with the Generalized Method of Moments.</a> PLoS One. 14 (1), 0197726 (2019).</li></ol>

The protocol can be used to measure the kinetics of any SSDC system, provided that the strand separation is observed during the experiment. The detection of cleavage is affected by observing the detachment of the tethered bead and therefore marks the instant of strand separation. All preceding steps occur before the detection of the cleavage; thus, only the total transit time is recorded.
The flow cell coverslip is functionalized via non-specific adsorption of antibody protein to the clean gla...

Site-specific DNA cleavage (SSDC) is a key step in many genomic transactions. For example, bacterial restriction-modification (RM)1 and CRISPR2 systems protect cells from attack by phages and plasmids by recognizing and cleaving foreign DNA at specific sequences. In type II RM, restriction endonucleases (REs) recognize short 4&#8211;8 base pair (bp) sequences via protein-nucleic acid interactions3. CRISPR-associated endonucleases, such as Cas9, bind to sites via hybridization of the target site with crRNAs bound to the endonucleases4. The creation of site-specific double stranded breaks (DSBs) are also the first step in many DNA recombination events5. For example, the diversity of antigen binding regions created by V(D)J recombination requires the recognition and cleavage of specific target sites6. Some transposons are known to target specific DNA sequences, as well7. Not surprisingly, many site-specific nucleases involved in these processes, such as Cas9, are a key component of gene editing technologies8. In addition, novel site-specific endonucleases (i.e., zinc finger nucleases9 and TALENS10) have also been engineered to edit genomes.
Many methods have been employed to measure the kinetics of site-specific cleavage of nucleic acids. These include gel analysis, fluorescence11,12, and sequencing based methods13. A major advancement was achieved with the tethering of microbeads, which allows DSBs in single molecules of DNA to be detected by the motion of a bead after strand separation. In these methods, different types of forces are employed to ensure strand separation and motion of the bead post-cleavage. In one case, optical traps have been used to measure cleavage of DNA by EcoRV14. In these experiments, target search is the objective of the investigation, with conditions optimized so that site-specific binding is the rate limiting step. One drawback of optical traps is that only a single DNA can be observed at a time. In addition, a periodic large pulling force has to be applied to test for the strand separation.
Another technique uses a combination of flow and weak magnetic forces to pull on the bead in a continuous manner15. In this way, diffusion limited cleavage by NdeI is measured. The method employed allows for the simultaneous measurement of several hundred DNAs at once, allowing for statistical significance to be attained in a single experiment. Experiments based on magnetic tweezers have also been used. In one such study, a retroviral integrase was studied by including a DSB in the insertion oligonucleotide16. Successful integration resulted in the incorporation of DSB in the tethered DNA and loss of the attached bead. In a similar study of the ATP-dependent type III restriction endonuclease EcoPI, tens of DNAs were observed in a single experiment17. Magnetic tweezers hold the advantage that tension, as well as DNA looping, can be controlled and monitored during the reaction.
Presented here is a highly parallel single molecule method for measuring SSDC kinetics, which takes advantage of recent improvements in large-scale tethering of DNAs. This method is an improvement and extension of previous methods used to measure DNA replication18, contour length of DNA19, and cleavage by REs15. In this technique, linear DNAs containing a single copy of the recognition sequence are prepared with biotin at one end and digoxigenin at the other. The biotin binds streptavidin, which is covalently attached to a paramagnetic microbead. The DNA-bead complexes are injected into a microfluidic channel that has been functionalized with anti-digoxigenin FAB fragments. The DNA then tethers to the surface attachment points via binding of the digoxigenin to the adsorbed FAB fragments. Weak magnetic forces applied with a permanent magnet keep the bead from sticking non-specifically to the surface. Samples can be injected rapidly (&#60;30 s) into the flow channel to activate the cleavage reaction. Flow is turned off during data collection. As each DNA is cleaved, the exact time of cleavage can be determined by recording the frame in which the bead moves up and out of the focal plan of the objective, thus disappearing from the video record. A frame-by-frame count of remaining beads can be used to quantify the reaction progress.
Presented below is the complete protocol as well as example data collected using NdeI. As an example of how the technique can be applied, cleavage rates for a range of protein concentrations are measured at two different concentrations of magnesium, an essential metal cofactor. Although this application of the protocol uses NdeI, the method can be adapted for use with any site-specific nuclease by varying the DNA substrate design.

<table>
	<tbody>
		<tr>
			<td>5 minute Epoxy</td>
			<td>Devcon</td>
			<td>14250</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-digoxigenin FAB fragments</td>
			<td>Roche Diagnostics</td>
			<td>11214667001</td>
			<td></td>
		</tr>
		<tr>
			<td>camera and software</td>
			<td>Jenoptik</td>
			<td>GRYPHAX SUBRA</td>
			<td></td>
		</tr>
		<tr>
			<td>data analysis software</td>
			<td>Vernier Inc.</td>
			<td>LP</td>
			<td></td>
		</tr>
		<tr>
			<td>double sided tape</td>
			<td>Grace Biolabs</td>
			<td>SA-S-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol 95%</td>
			<td>VWR</td>
			<td>89370-082</td>
			<td></td>
		</tr>
		<tr>
			<td>forward primer: digoxigenin-CCAACTTAATCGCCTTGC</td>
			<td>Integrated DNA Technologies</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>image analysis software</td>
			<td>National Institutes of Health</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Nikon</td>
			<td>TE2000</td>
			<td></td>
		</tr>
		<tr>
			<td>knife printer</td>
			<td>Silhouette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M13mp18 DNA</td>
			<td>New England Biolabs</td>
			<td>N4040S</td>
			<td></td>
		</tr>
		<tr>
			<td>MyOne streptavidin beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>65601</td>
			<td></td>
		</tr>
		<tr>
			<td>NdeI enzyme</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR cleanup kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>pluronic F-127</td>
			<td>Anatrace</td>
			<td>P305</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylene tubing PE-20</td>
			<td>BD Intramedic</td>
			<td>427406</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylene tubing PE-60</td>
			<td>BD Intramedic</td>
			<td>427416</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 Mastermix</td>
			<td>New England Biolabs</td>
			<td>M0492S</td>
			<td></td>
		</tr>
		<tr>
			<td>rare earth magnet 0.5&#34; OD 0.25&#34; ID</td>
			<td>National Imports</td>
			<td>NSN0814</td>
			<td></td>
		</tr>
		<tr>
			<td>rare earth magnet 0.75&#34; OD 0.5&#34; ID</td>
			<td>National Imports</td>
			<td>NSN0615</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer: biotin-TGACCATTAGATACATTTCGC</td>
			<td>Integrated DNA Technologies</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>Kent Scientific</td>
			<td>Genie Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Casein from bovine Milk</td>
			<td>Sigma-Aldrich</td>
			<td>C6905</td>
			<td></td>
		</tr>
	</tbody>
</table>

parallel high throughput single molecule kinetic assay for site-specific dna cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

Using this technique, the SSDC rates of NdeI were measured for a range of protein concentrations (0.25&#8211;4.00 U/mL) at two different concentrations of magnesium (2 mM and 4 mM). Each of these conditions was replicated at least twice, with a few hundred to 1,000 tethered DNAs per experiment. Figure 2 describes the experimental design. Figure 3 shows examples of data collection and analysis details. Figure 4 illustrates how the ra...

Watch this Scientific Journal Video about Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage at JoVE.com

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

This protocol can be applied to any process that results in double stranded DNA breaks, yields detailed quantitative data on kinetics. Although this method has single molecule resolution, it measures up to 1000 DNAs in a single experiment, thus providing good statistics. The primary application of this method is to study the mechanisms involved in DNA binding and modification.For example, we are interested in studying DNA target search, which is relevant to all DNA binding proteins. When attempting this technique for the first time, remember that single molecule tethers are delicate, and once form, must be handled with care. Proper proportion of functionalized surfaces is essential.There are a few tricks for making the flow so. As well as switching sample tubes during data collection, visual demonstration can help someone who is new to this procedure. To wash the cover slips, place them in staining jars and sonicate them with ethanol for 30 minutes.Thoroughly rinse them with deionized and distilled water, then immerse them in one molar potassium hydroxide and sonicate them for 30 more minutes. Repeat the wash sequence, making sure to rinse the cover slips with water after each wash. Store the cleaned cover slips in staining jars with water.Use a clean razor to cut eight centimeter long loading and exit tubes, and insert them into holes in a clean glass slide. Epoxy the tubing and let cure for five minutes to secure it. Apply double sided tape pre-cut with the channel pattern to the glass slides, and smooth it out with plastic forceps to achieve a good seal.Peel the backing off the tape, and apply a clean cover slip, smoothing it out with the forceps. Then, epoxy the edge of the cover slip to seal the flow cell and let it cure. Prepare the labeled DNA for tethering by performing PCR according to manuscript directions.Then purify the PCR product with a PCR cleanup kit. Prepare 10 milliliters of buffer A, and degas it in a vacuum desiccator for at least one hour. To functionalize the flow cell, inject 25 microliters of anti-digoxigenin and fab fragments in PBS, into the flow channel, using a gel loading tip to fit into the PE60 tubing.Incubate the flow cell at room temperature for 30 minutes. After the incubation, pull 0.5 milliliters of buffer A through the channel with a syringe, taking care to not introduce air into the channel. Then, mountain the flow cell on an inverted microscope.Hook up the outlet tube to a syringe pump, and put the inlet tube into a micro centrifuge tube containing buffer A.Manually pull at least 0.5 milliliters of buffer A to flush the system and prime the pump. Then let the pump run at 10 microliters per minute for at least five minutes to equilibrate the system. Vortex the stock bottle of beads, and pipette 1.6 microliters of the beads into 50 microliters of buffer A, then vortex them again.Place the beads on a magnetic separator and pipette out the buffer. Re-suspend them in 50 microliters of buffer A and vortex them. After the last wash, re-suspend the beads in 100 microliters of buffer A for a final concentration of 160 micrograms per milliliter.Prepare 480 microliters of 0.5 picomolar labeled DNA substrate in buffer A, and pipette 20 microliters of bead suspension into the diluted DNA. Place the mixture on a rotator for three minutes. After the three minutes, immediately load the sample into the channel at a flow rate of 10 microliters per minute for 15 minutes, or until sufficient bead tethering is observed.Wash the channel of all free beads by switching the inlet tube to a fresh tube of buffer A, and flowing it in at 50 microliters per minute for at least 10 minutes or until no loose beads are observed. And it's helpful to use an inline valve to cut off flow and switching the tubing. Switching tubing in one smooth motion and make sure liquid levels in new and old sample tubes match to avoid backflow.Before collecting data, prepare the correct concentration of DNA cleavage enzyme, and insert the inlet tube into the sample. Lower the magnet over the flow cell. Set the pump to inject 80 microliters of sample at 150 microliters per minute and begin data collection.One minute into data collection, activate the pump. Shown here is a representative image of the tethered beads before and after the reaction. Data analysis was performed to quantify the number of beads remaining tethered throughout the cleavage reaction.This technique was used to measure the site specific DNA cleavage rates of Nde1 for protein concentrations ranging from 0.25 to four units per milliliter, and two different concentrations of magnesium. Each condition was replicated at least twice, with a few 100 to 1000 tethered DNA is per experiment. At sufficiently low protein concentrations, the rate is proportional to protein and independent of magnesium.For high protein concentrations, the rate is dependent on magnesium, but independent of protein concentration. When attempting this protocol, keep in mind that air bubbles in the tubing and flow channel can run the experiment. Make sure not to allow any air into the lines while switching tubing, and degas all buffers before using.The tethering method allows one to explore the effect of tension in the DNA on the reaction. This can be achieved by varying the magnet strength or applying flow during data collection.

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