Team:Valencia UPV/Hardware/Manual

User manuals

Index

Handbook for HYPE-IT editing

Here we show detailed instructions of how to edit a plant using the HYPE-IT system based on Cas9. The process is explained, with links to the specific protocols.

Target search

Log in the Data Processing Software’s Web http://hypeit.cloudno.de/

A) Unknown target

  1. Go to “Target Search (DDBB)” to access the search engine.
  2. Search your phenotypic trait of interest to know the gene to edit. You can choose it filling up three fields: plant name or species, phenotypic characteristic and/or the name of the gene.
  3. You will be provided with a list of possible options related to your search. To see details of your choice, click on the left icon.
  4. Here you have all the information about the target gene. Click on “Show more” to access the Data Processing Software and find the optimal gRNA for your target. Move to step 5.


B) Known target

  1. Go to “Find optimal gRNA” to access the Data Processing Software.
  2. Paste the DNA sequence of the known gene in FASTA format and click on “Find Targets”


C) In both cases

  1. The software shows a list of possible gRNAs with their score. Select the highest one and you will be provided with the optimal gRNA. Additionally, the software provides the primers to amplify the target of your plant with the overhangs to use them with the Goldenbraid System with the HYPE-IT strategy.
  2. Order to synthesize to any synthesis company of your choice the two primer oligonucleotides (DNA sequences):
    • Primers + Goldenbraid overhangs Forward
    • Primers + Goldenbraid overhangs Reverse
  4. The gRNA is synthetized after step 6 of the gRNA testing system protocol.
    • RNA + Goldenbraid overhangs Forward
    • gRNA + Goldenbraid overhangs Reverse
    • Target consensus + Goldenbraid overhangs Forward
    • Target consensus + Goldenbraid overhangs Reverse
    • Target knockout + Goldenbraid overhangs Forward
    • Target knockout + Goldenbraid overhangs Reverse


These overhangs will allow to insert the amplified gene fragment of your variety in our gRNA Testing System. The gRNA Goldenbraid overhangs are used to insert the gRNA in a plasmid based in Goldenbraid for plant infection.

Identification of the target

  1. Perform a genomic DNA extraction of the plant. The protocol used will depend on the plant and variety that you use. As an example, you can check here the protocols we have used for orange and rice extraction.
  2. Run electrophoresis gel (agarose 1%) of the DNA extraction following this protocol. You should see a band higher than the molecular weight ladder (above 10kb).
  3. Perform a PCR in the thermal cycler of the genomic DNA with the ordered primers with Goldenbraid overhangs in order to obtain the fragment of the target gene. Depending on the primers given by the Software and the polymerase you use, the hybridization temperature for the PCR will be different. You can use an online calculator to know this temperature. It is usually provided by the company where the primers were bought. When using the HYPE-IT thermal cycler, it should be used mineral oil to cover the liquid, in order to avoid evaporation.
  4. Run electrophoresis gel (agarose 1%) of the PCR products following this protocol. You should see a band lower than 100kb.
  5. Send the PCR product to sequence to a sequencing service. The primers you can use to sequence are the same used to amplify the gene fragment.
  6. Once you know the sequence of the gene you want to edit, you have two possible paths to follow:
    • If the sequence is the same that the gRNA chosen in the Data Processing Software, you can order to synthetize the gRNA + Goldenbraid overhangs shown. Additionally, you will need to synthetize the target consensus + Goldenbraid overhangs sequences.
    • If the sequence is different that the gRNA chosen in the Data Processing Software, you can either choose a different gRNA in the software (repeating everything from step 1) or you can order to synthetize your own sequence adding these overhangs:
      • ATTG + sequence: gRNA forward
      • AAAC + sequence (reverse complementary): gRNA reverse
      • AATG + sequence: target consensus forward
      • CGAA + sequence (reverse complementary): target consensus reverse.
      • AATG + sequence removing the 16th nucleotide: target knockout forward
      • CGAA + sequence removing the 16th nucleotide (reverse complementary): target knockout reverse.
  8. To obtain the functional gRNA, put in an eppendorf the gRNA forward and gRNA reverse oligonucleotides ordered in step 6. It is necessary to put 18μL of water and 1μL of each oligonucleotide. Leave 30 minutes at room temperature.
  9. Make a ligation reaction with the gRNA obtained in step 6, U6 promoter and psgRNA (both provided in the reagents kit). Follow the ligation protocol to ligate in α1. You will obtain the device U6:gRNA:psgRNA. Now you can directly edit your plant or you can test if you wish the efficiency of the gRNA in our gRNA testing system.


gRNA testing system

  1. Ligation using Goldenbraid assembly system to obtain the complete gRNA testing system device. The ligation is made following the ligation protocol. The plasmids, linker and promoters are provided in the reagents kit. The ligations to be made are:
    • In α1 plasmid: 35s or 35s:5’ + target consensus + linker (BBa_K2017003, BBa_K2017004, BBa_K2017005 or BBa_K2017006) + Tnos.
    • In α1 plasmid: 35s or 35s:5’ + target knockout + linker (BBa_K2017003, BBa_K2017004, BBa_K2017005 or BBa_K2017006) + Tnos.
  3. Transform electrocompetent E. coli with the ligation products of step 1 following the transformation protocol.
  4. Culture E. coli in a Petri dish with kanamycin resistance, as stated by petri dish culture protocol.
  5. After waiting the time specified in the protocol in point 3, pick white colonies in the Petri dish and make a liquid culture.
  6. Perform a miniprep to extract the plasmid of E. coli and check if the selected colonies have the desired plasmid. We use the E.Z.N.A ® ®. Plasmid Mini Kit I, whose instructions are explained in the protocols
  7. Digestion of minipreps with EcoRI (specific enzyme for α1 digestion). Here you can find the digestion protocol
  8. Run electrophoresis gel (agarose 1%) of the digestion products. You should see two bands, one at 6kb and other between 3 and 4kb. Steps 6 and 7 are made to check if the plasmid in the picked colonies is the plasmid ligated in step 1.
  9. To obtain the functional target consensus, put in an eppendorf the target consensus forward and target consensus reverse oligonucleotides ordered in step 6 of “Identification of the target” section. It is necessary to put 18μL of water and 1μL of each oligonucleotide. Leave 30 minutes at room temperature.
  10. Repeat the same as in step 8 to obtain the target knockout, mixing target knockout forward and target knockout reverse.
  11. As our experiments showed that the system was more efficient in cis (Cas9 and gRNA in the same plasmid as the testing system). So, ligate the U6:gRNA:psgRNA with the plasmids obtained in step 5 (testing system target consensus and testing system target knockout), in Ω2 plasmid, as in ligation protocol. Each testing system must be ligated separately with the U6:gRNA:psgRNA (only target consensus) and with U6:XT1gRNA:psgRNA.
  12. Transform electrocompetent E. coli with the ligation products of step 10 following the transformation protocol.
  13. Repeat steps 3 to 7 for the transformed E. coli in step 11.
  14. Ligate the plasmids obtained in step 10 with the 35s:Cas9:Tnos + SF (provided in the reagents kit), in an α1 plasmid. Repeat steps 11 and 12.
  15. Transform Agrobacterium C58 with the extracted plasmids in the minipreps. Repeat the same protocol as E. coli transformation, but using 1440V. You should have three plasmids:
    • Cas9 + gRNA testing system with Target consensus + gRNA
    • Cas9 + gRNA testing system with Target consensus + XT1gRNA
    • Cas9 + gRNA testing system with Target knockout + XT1gRNA
  17. Repeat steps 3 to 7 for the transformed Agrobacterium in step 14.
  18. Agroinfiltrate Nicotiana benthamiana with the three different Agrobacterium obtained in step 15. Follow the Agroinfiltration protocol. The experimental design is of your choice. We suggest to make the following experiments (the devices not created by you are provided in the reagents kit). Each point represents one plant to be agroinfiltrated with the stated agrobacterium cultures.
    • 35s:Luciferase:Tnos (positive control)
    • pNOS:Luciferase:Tnos (negative control)
    • Cas9:gRNA testing system with Target consensus:gRNA + 35:Renilla:Tnos (sample)
    • Cas9:gRNA testing system with Target consensus: XT1gRNA + 35:Renilla:Tnos (negative control)
    • Cas9:gRNA testing system with Target knockout:XT1gRNA + 35:Renilla:Tnos (positive control)
  20. Three days post-infiltration, perform a luciferase assay to test the efficiency of the gRNA you chose in the database or you designed after sequencing. It is recommended to take at least three replicates per agroinfiltrated leaf. You can follow the luciferase assay protocol here.


Plant editing

  1. It is going to be used the U6:gRNA:psgRNA device obtained in step 8 of “Identification of the target” section. However, instead of using the overhang ATTG in the forward brand, it will be used the overhang AGCC.
  2. Ligate the U6:gRNA:psgRNA with the TMV split-Cas9 vector (provided in the reagents kit). Follow the ligation protocol.
  3. Transform electrocompetent E. coli with the ligation products of step 1 following the transformation protocol.
  4. Culture E. coli in a Petri dish with kanamycin resistance, as stated by petri dish culture protocol.
  5. After waiting the time specified in the protocol in point 3, pick white colonies in the Petri dish and make a liquid culture.
  6. Perform a miniprep to extract the plasmid of E. coli and check if the selected colonies have the desired plasmid. We use the E.Z.N.A ® ®. Plasmid Mini Kit I, whose instructions are explained in the protocols
  7. Digestion of minipreps with EcoRI (specific enzyme for α1 digestion). Here you can find the digestion protocol
  8. Run electrophoresis gel (agarose 1%) of the digestion products. You should see two bands, one at 6kb and other between 3 and 4kb. Steps 6 and 7 are made to check if the plasmid in the picked colonies is the plasmid ligated in step 2.
  9. Transform the plasmid obtained in step 6 in Agrobacterium, following the same steps from 3 to 7.
  10. Agroinfiltrate Nicotiana benthamiana with split-Cas9 + gRNA in TMV and split-Cas9 in PVX (provided in the reagents kit). Follow the agroinfiltration protocol. Take the necessary preventive measures to avoid contact of the agroinfiltrated plants with other plants. The viral systems are safe and do not have the coat protein, but it is still advisable to manage it with caution.


Modified plant obtention

  1. Five days post-agroinfiltration, make micropropagation of callus obtained from an infected leave of the plant.
  2. Make in vitro culture of the callus in a phytotron to grow a plant with its cells edited. The grown plant will have the knockout.


Labware User Manual

The labware created by HYPE-IT have multiple tools, in this manual it will be explained how to operate them.
All the tools, except the phytotron, are controlled through the LCD display and the rotary encoder.
A interface with options will be display in the screen. By turning the rotary encoder it is possible to change the selected option. When the desired option is selected, push the rotary encoder in order to access to that option.

Electroporator

Place an electroporator cuvette in the electroporator. Make sure that the electrodes of the cuvette contact with the terminal of the electroporator.
Choose the option of “Electroporator” in the principal menu in the menu, then the options of the electroporator will be displayed:
“Set voltage”: this option is used to set the voltage of electroporation. The typical value is 1500 volts.
“Set time”: this option is used to set the duration of the electroporation in milliseconds. The typical value of this parameter is 5 milliseconds.
“Run”: this option will start the electroporation. When finished it will be displayed the real voltage of electroporation.
“Go back”: this option will change the interface to the principal menu.

Colorimeter

Place a cuvette in the colorimeter in the right way. Avoid touching the walls of the cuvette in order to have better measures.
Choose the option of “Colorimeter” in the principal menu in the menu, then the options of the colorimeter will be displayed:
“Blank”: this option is used to do the blank. This is always needed to do before start doing measures. A cuvette with the solvent has to be placed in order to do the blank.
“Measure OD600”: this option is used to do the measure of optical density. When the measure is done it will be displayed the value of the optical density.
“Go back”: this option will change the interface to the principal menu.

Centrifuge

Place the eppendorf tubes in the rotor of the centrifuge. Do not desbalance the rotor of the centrifuge, this is extremely dangerous.
Choose the option of “Centrifuge” in the principal menu in the menu, then the options of the centrifuge will be displayed:
“Set RPM”: this option is used the set the speed in rpm. The max rpm is 13.000.
“Set time”: this option is used set the duration in minutes will be activated the centrifuge.
“Status”: this option will shown in the screen the actual speed and remaining time.
“Run”: this option will start the centrifuge.
“Go back”: this option will change the interface to the principal menu.

Gel electrophoresis unit

Connect the electrophoresis cuvette to the terminals in the labcase, make sure to respect the electrical polarity.
Choose the option of “Electrophoresis” in the principal menu in the menu, then the options of the electrophoresis will be displayed:
“Set voltage”: this option is used to set the voltage of the electrophoresis in volts. The typical value of this parameter is 100 volts.
“Set time”: this option is used to set the duration of the electrophoresis in minutes. The typical value of this parameter is 30 minutes.
“Run”: this option will start the electrophoresis. The remaining time of the electrophoresis will be displayed in the screen. When finished the electrophoresis a message will se shown in the screen.
“Go back”: this option will change the interface to the principal menu.

Stirrer with temperature control

Place the sample tubes in the stirrer.
Choose the option of “Stirrer” in the principal menu in the menu, then the options of the stirrer will be displayed:
“Set temperature”: this option is used to set the temperature of the stirrer in degrees. The typical value of this parameter is 37 degrees.
“Run”: this option will begin to shake the stirrer and start the temperature control.
“Stop”: this option will stop the shaking of stirrer and stop the temperature control.
“Status”: this option will show the actual temperature of the stirrer.
“Go back”: this option will change the interface to the principal menu.

Thermal cycler

Place the reaction tubes in the thermocycler. Avoid touching the metallic surface, it might be hot.
94 62 72
Choose the option of “Thermal Cycler” in the principal menu in the menu, then the options of the thermocycler will be displayed:
“Set T1”: this option is used to set the temperature in degrees of the first denaturalization. The typical value of this parameter is 94 degrees.
“Set time1”: this option is used to set the duration in seconds of the first denaturalization. The typical value of this parameter is 600 seconds.
“Set T2”: this option is used to set the temperature in degrees of the denaturalization. The typical value of this parameter is 94 degrees.
“Set time2”: this option is used to set the duration in seconds of the denaturalization. The typical value of this parameter is 30 seconds.
“Set T3”: this option is used to set the temperature in degrees of the annealing. The typical value of this parameter is 62 degrees.
“Set time3”: this option is used to set the duration in seconds of the annealing. The typical value of this parameter is 30 seconds.
“Set T4”: this option is used to set the temperature in degrees of the elongation. The typical value of this parameter is 62 degrees.
“Set time4”: this option is used to set the duration in seconds of the elongation. The typical value of this parameter depends on the size of the amplified DNA fragment.
“Set T5”: this option is used to set the temperature in degrees of the hold. The typical value of this parameter is 4 degrees. This temperature is used to keep the PCR when it have finished.
“Run”: this option will begin the PCR.
“Status”: this option will show the actual cycle of the PCR and the actual temperature.
“Go back”: this option will change the interface to the principal menu.

Luminometer

Place the three sample tubes in the luminometer and make sure the cover is well placed. The idea about the three sample tubes is to measure at the same time the positive and negative control and the sample.
Choose the option of “Luminometer” in the principal menu in the menu, then the options of the luminometer will be displayed:
“Measure”: this option is used to do the measure of light intensity When the measure is done it will be displayed the light measured in the three samples.
“Go back”: this option will change the interface to the principal menu.

Phytotron

The phytotron does not have a physical interface so to change the parameters it is necessary to reprogram the Arduino.
In the Arduino code of the phytotron is explained how to change parameters as the temperature reference or the duration of the light cycles.

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