The bacterial CRISPR-Cas9 system has substantially increased experimental options for life scientists. However, several CRISPR approaches depend on the simultaneous delivery of multiple guide RNAs into individual cells. String assembly gRNA cloning allows the simple and fast generation of multiplex gRNA expression vectors in one single cloning step.
But STAgR is not only simple, it is also efficient, cheap, and easy to customize. To begin, add the N20 gRNA sequences to the forward amplification primers for STAgR DNA string as overhangs. Add sense gRNA sequences five prime to the forward primer, Scaffold forward primer, for a conventional scaffold, or to SAM forward primer for an MS2-containing scaffold.
Next, add the reverse complement N20 gRNA sequences to the reverse primer sequences, choosing RP primers, depending on the specific promoters and strings used. To begin generating the individual cloning fragments for Gibson Assembly, transfer 10 microliters of the high-fidelity buffer to a tube. Add one microliter of 10 milimolar dNTPs and 0.25 microliters of both 100 micromolar overhang primers.
Add 10 nanograms of the DNA template string, or vector, and 1.5 microliters of DMSO. Add enough water to bring the final volume to 49.5 microliters, and then add 0.5 microliters of HF polymerase. Incubate the reactions on a thermocycler, as outlined in the text protocol.
After this, take a 5.5 microliter aliquot from the PCR reaction. Add loading dye to all aliquots, and load it on a 1%agarose gel. Load an appropriate DNA ladder for sizing, and run the gel in an appropriate gel-running buffer at 120 volts for 30 minutes.
While the gel is running, add five microliters of the buffer provided with the restriction enzyme, and 0.5 microliters DpnI to the remaining 44.5 microliters of vector PCR reaction to remove residual plasmid template used during the PCR. Incubate at 37 degrees Celsius for 30 to 60 minutes. Check to make sure that the amplicons are the correct size.
To begin DNA purification, add 1.8 microliters of magnetic beads per one microliter of PCR product, and mix by pipetting up and down. Incubate at room temperature for two minutes. Using a magnet, separate the beads in DNA fragments from the residual liquid.
Rinse the pellet with 70%ethanol without fully resuspending it, to wash the beads. Repeat this wash one additional time. Using a pipette, remove all of the ethanol and let the pellet air dry.
Then add 15 to 20 microliters of water, and pipette up and down to mix and dissolve the pellet. Use a magnet to separate the beads from the liquid. Transfer the clear supernatant to a new tube.
After this, use a spectrophotometer to determine the DNA concentrations. First, prepare the 5x isothermal reaction buffer as detailed in the text protocol. For the Gibson Assembly Master Mix, combine 320 microliters of the 5x isothermal reaction buffer with 697 microliters of water.
Add 3 microliters of T5 exonuclease, 20 microliters of DNA polymerase, and 160 mciroliters of Taq DNA ligase. Transfer 7.5 microliters of the Assembly Master Mix to a fresh tube. Then add 2.5 microliters of insert in vector, as outlined in the text protocol.
Incubate the samples at 50 degrees Celsius for 45 to 60 minutes. After this, store samples on ice, or at 20 degrees Celsius, for subsequent transformation. When ready to transform the bacteria, thaw Chemically Competent TOP10 E.Coli bacteria on ice.
Next, add five microliters of the Gibson Mix to 50 microliters of competent bacteria, and mix by gently flicking the bottom of the tube. Incubate on ice for 30 minutes. Place the tube in a 42 degree Celsius water bath or heat block for 45 seconds to heat-shock the bacteria.
Then, put the tubes back on ice. Add 250 microliters of SOC medium to the bacteria, and let them recover at 37 degrees Celsius in a shaking incubator for 30 to 45 minutes. After the bacteria have recovered, plate them onto 1.5%LB Agar plates that contain 100 micrograms per milliliter ampicillin.
Incubate the plates at 37 degrees Celsius over night. To begin, prepare at least two sets of 200 microliter PCR reaction tubes for each construct. Fill one of the sets of reaction tubes with 100 microliters LB medium, containing 100 microgram per milliliter of ampicillin.
Using a sterile pipette tip, scratch off the biological material from one bacterial colony, and spread it at the bottom of an empty 200 microliter PCR reaction tube. Immediately transfer the pipette tip to the second corresponding reaction tube that contains LB medium. Swirl the tip around to make sure that some of the bacteria are transferred to the LB media.
Incubate at 37 degrees Celsius for later use. Then, prepare 10 microliters of PCR Master Mix per reaction tube, as outlined in the text protocol. Add 10 microliters of the PCR Master Mix to the labeled PCR reaction tubes, without the LB media.
Using a thermocycler, incubate the reactions as outlined in the text protocol. After this, add loading dye to the amplified fragments, and load them on a 1%agarose gel. Load an appropriate DNA ladder for sizing, and run the gel in an appropriate gel-running buffer at 120 volts for 30 minutes.
Calculate the theoretical size of the amplicon 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. Using the corresponding 100 microliter culture, inoculate a 2.5 milliliter overnight LB culture containing 100 microgram per milliliter ampicillin.
Incubate at 37 degrees Celsius for 12 hours. Then, use a commercial plasmid mini-kit to extract the plasmid DNA, according to the manufacturer's instructions. In this procedure, string assembly gRNA cloning is used to quickly generate gRNA expression plasmids.
A typical outcome of the PCRs used to obtain the STAgR pieces is seen here. The six amplicons represent linear DNA pieces, each containing the individual gRNA N20 sequences on their ends. The plasmid backbone is extended with the targeting sequences of gRNA1 and gRNA6, and therefore possesses the required overlaps to two other PCRs for Gibson Assembly.
After purification, a DNA yield of at least one microgram for vectors and inserts can be achieved. After Gibson Assembly, bacterial transformation results in 100 to 700 bacterial colonies. Representative analysis of 22 bacterial colonies, via colony PCR following a STAgR protocol with six gRNA cassettes, indicates that seven clones show the expected amplicon size for full assembly.
The other clones likely received STAgR vectors, containing one to five gRNA cassettes, whereas one clone is completely empty. Once mastered, this technique can be done in a few hours. Performed properly, it allows the generation of a multitude of multiplex gRNA expression vectors in less than a working week.
While attempting this procedure, it's important to pay attention to the correct assign and combination of N20 overhang primers. After watching this video, you should have a good understanding on how to create your own multiplex guide RNA expression vectors.
