The overall goal of this cloning and transformation procedure is to efficiently generate CRISPR Vectors and knock out mutant tomato roots that can be used in functional genomic studies. This method is a useful tool in the field of plant genomics because it can generate a large number of knockout mutants that can be used in a variety of root processes. The main advantage of this technique is that the cloning procedure can be accomplished in a single step, entrenching that group materials can be obtained in just a matter of weeks.
First, design guide RNA or gRNA oligos to include the GN19 portion of the target motifs flanked by the five prime and three prime 20 nucleotide sequences required for DNA assembly. Digest one to five micro grams of P201N caznine plasmid with the restriction enzyme Spe1 at 37 degrees Celsius for two hours. After column purifying the digest according to manufacturer's instructions, re-suspend in 15 microliters of 10 millimolar Trs HCL.
Quantify the amount of DNA by UV Spectrophotometry. Perform a second digest with a restriction enzyme Swa1 at 25 degrees Celsius for two hours. Check 100 to 200 nanograms on a zero point eight percent agarose gel to confirm complete digestion.
A correctly digested plasmid will have a single band at 14, 313 base pairs. To PCR amplify the medicago truncatula use six promoter and scaffold DNA's from the puck gRNA shuttle plasmid. Use the primer Swa1 and Mtu6F and MTU6R, and scaffold F in Spe1 scaffold R, in a high fidelity polymerase.
Visualize three microliter aliquats of PCR products on a one percent agarose gel to confirm amplification. The MTU6 amplicon is 377 base pairs, and the scaffold amplicon is 106 base pairs. Column purify the remaining PCR products according to manufacturer's instructions.
Quantify by UV Spectophotometry as before and store at negative 20 degrees Celsius. Next, re-suspend the entire tube of gRNA oligos to 100 micromolar in laboratory grade water. Add one microliter of the oligos to 500 microliters of 1xNEB buffer two point one and mix well.
Program a thermal cycler with a heated lid to hold at 50 degrees Celsius. Combine 100 nanograms of the linearized vector, 50 nanograms of MtU6 promoter, 12 nanograms of Scaffold, and one microliter of diluted gRNA oligo in water to a final volume of five microliters. Add five microliters of 2X high fidelity DNA Assembly Mastermix.
Mix well and spin down. Incubate the reaction for 60 minutes in the 50 degree Celsius thermocycler. Following the hour at 50 degrees Celsius, place the reaction on ice and then use two microliters to transform competent E.Coli cells using standard techniques.
Plate the transformation on LB Agar supplemented with 50 milligrams per milliliter Kanamycin and incubate at 37 degrees Celsius overnight. Screen the colonies using PCR with the primer StUb3p 218R and 1Sce1R. Dilute an alequat of the remaining DNA assembly reaction with water one to five, and use one microliter as a positive control for the colony screen.
Also include one nanogram of circular P201N casnine plasmid as a no insert control. After PCR amplification using the parameters contained in the written portion of the protocol, visualize PCR product on a one percent agarose gel. Correct insertions have a banded 725 base pairs and vectors without inserts will have a 310 base pair band.
Grow positive colonies in LB Can50 liquid cultures overnight at 37 degrees Celsius. The next day, purify plasmids using a plasmid purification kit according to manufacturer's instructions. After Sanger sequencing the purified plasmids with the StuB3P218R primer, align chromatograms to the MtU6 promoter, Target, and scaffold sequences to ensure no errors were introduced during cloning.
Perform a diagnostic digest by digesting one microgram of plasmid with EcoR5 and Sti1. When visualized on a zero point eight agarose gel, this banding patter should be seen. Finally, transform the plasmids into Agrobacterium rhizogenes strain ARqua1 by adding one microliter of the plasmid prep to 50 microliters of electrocompetent cells and electroporating in a one millimiter cuvette.
Add approximately 500 microliters of SOC Media and shake at 28 degrees Celsius for two hours. Then plate on LB Can50 plates and grow at 28 degrees Celsius for two days. Begin this section of the procedure by sterilizing tomato seeds in 20 percent household bleach for 15 minutes with constant mixing.
Then transfer the seeds to a laminar flow hood. Remove the bleach and wash in sterile laboratory grade water three times. Plate 30 seeds in a GA7 box containing one half MS Media.
Allow to germinate for around two days in the dark. Once the seeds have germinated, move the GA7 boxes into the light The day before transformation, streak out A.rhizogenes cultures on solid LB containing Can50 and grow at 28 degrees Celsius overnight. On the day of transformation, work in the laminar flow hood to add 25 microliters of Acetosyringone to 50 milliliters of one half MS and pour six milliliters into each culture tube.
Use a bent 200 microliter tip to scrape A.rhizogenes cells from the plate and resuspend in the six milliliters of one half MS liquid. Vortex the tube to completely resuspend the cells. After resuspending cells from each vector, take one milliliter of cells and measure the optical density at a fixed wavelength of 600 nanometers.
Add two milliliters of one half 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 most distal one centimeter off of the cotyledons, resulting in cotyledon pieces with two cut ends.
Add the ex plants to A.rhizogenes solutions and mix. Incubate for 20 minutes with occasional inverting. During the A.rhizogenes incubation, add a piece of filter paper to a petri dish for each construct transform.
Also, set one half MS solid media without antibiotics in the laminar flow hood to dry. Use sterile forceps to scoop cotyledons out of A.rhizogenes solution and place on dry filter paper. Cover with a petri dish lid to ensure tissues do not dry out while proceeding to the next transformation.
Next, blot cotyledons on the filter paper and transfer abaxial side up to one half MS media. Wrap the plates with surgical tape and co-cultivate in the dark at room temperature for two days. After co-cultivation, transfer cotyledons abaxial side up into supplemented one half MS medium.
Wrap with surgical tape. Maintain cultures under fluorescent lights at room temperature with a 16 hour photo period. After one point five to two weeks, use sterile forceps and a scalpel to excise roots at least two centimeters in length from the cotyledons and transfer to Ticarcillon/Clavulanate Acid and Kanamycin supplemented one half MS media.
Transfer ten to 15 roots to a single plate. Mark the position of the root tips with a marker. Wrap with surgical tape and maintain in the culture room.
After one week, transform roots are seen growing on the selective media. Harvest a subsample of transformed roots for DNA extraction using the preferred DNA extraction method. Hairy roots can be seen emerging from cotyledons 11 days after transformation.
The roots are selected on Ticarcillon/Clavulanate Acid and Kanamycin supplemented one half MS Media for approximately one week. Grey bars denote the position of root tips at the time of plating. This is an example of cloning and sequencing of PCR products to determine DNA mutations with two different gene targets in four different hairy root events.
The grey box denotes GN20 GG target sequence and delta indicates the type of mutation. The number of clones with the indicated mutation is shown at the right. This is an example of a polyacrylamide gel to determine DNA mutations.
Large deletions and heterduplex bands indicate the presence of DNA mutations at the target sequences. Six of 12 independent events were scored as mutants as indicated by the delta symbol. WT indicates the wild type control and NT the no template control.
Once mastered, tens to hundreds of CRISPR vectors can be produced in a single week and trendulate group materials can be obtained in a matter of weeks. While attempting this procedure, it's important to remove and inhibit the growth of any residual Agrobacterium. Overgrowth of Agrobacterium will inhibit and kill any root material.
After watching this video, you should have a good understanding of how to generate CRISPR vectors using DNA assembly, and how to test those vectors using a hairy root model system and tomato.
