This method allows for ease of creation of guide RNA expression plasmids for CRISPR-facilitated experiments. The main advantage of this technique is that cloning can be undertaken in a single step and paired guide RNAs can be created using single-guide RNA expression plasmids. To begin this protocol, dilute the lyophilized primers in 1X TE buffer to a final concentration of 100 micromolar.
Aliquot an equal amount of forward and reverse primers into PCR strip cap tubes. Vortex to mix. Next, spin down the guide RNA oligonucleotide mixtures at 100 times G for 15 seconds.
Incubate the reaction at room temperature for five minutes before setting up the ligation. Add between one and five micrograms of the selected PSB700 guide expression vector to BSNB1 using 0.5 microliters of BSNB1 per one microgram of vector. Add distilled water until the total volume is 40 microliters and conduct the digestion for one hour at 55 degrees Celsius.
Run the digestion products on a 1.5%low melting agarose gel. Cut out the digested vector backbone band that corresponds to a fragment approximately 9 kilobases in size. Transfer this gel slice to a 1.5 milliliter microcentrifuge tube.
Using a commercial gel-purification kit, extract the DNA from the agarose gel according to the manufacturer's instructions. Then dilute the DNA into 10-15 microliters of TE buffer to obtain a concentrated elute. When ready to perform ligation add 15 microliters of distilled water to a vial.
Add 1 microliter of the previously annealed guide RNA oligonucleotides, one microliter of the BSNB1 digested PSB700 guide expression vector and 2 microliters of 10XT4 DNA ligase reaction buffer. Then mix this solution by vortexing. Add one microliter of DNA ligase and vortex again.
Spin down the solution at 100 times G for 15 seconds. Incubate the reactions at room temperature over night making sure to include a no insert negative control reaction that has a BSNB1 digested vector alone without an annealed guide RNA oligote insert. To begin, remove E.coli from storage at minus 80 degrees Celsius and thaw it on ice for 5 to 10 minutes.
Add 0.5 microliters of the prepared reaction mixture to eight microliters of competent E.coli. Keep the mixture on ice for 30 minutes. Heat-shock the mixture at 42 degrees Celsius for 45 seconds.
Then let the mixture rest on ice for two minutes. Using a rotary shaker, recover the culture in 250 microliters of SOC media using the conditions listed here for NEB DH5a or NEB Stables. After this, plate 80 microliters of the culture on an appropriate antibiotic resistance lysogeny broth plate.
Incubate overnight at 37 degrees Celsius for NEB DH5a or at 30 degrees Celsius for NEB Stables. To begin, use the Phusion GC special PCR protocol to create the needed fragment as outlined in the text protocol. Run this PCR product on a 1%agarose gel and verify that one band is seen at approximately 490 base pairs.
Using a gel extraction kit cut and extract this PCR product. Then aliquot a prepared 1X master mix into PCR tubes. Using a multi-channel pipette to add one microliter of the PCR product at a concentration of 40 femtomoles per microliter.
In one microliter of the PSB700 vector add a concentration of 40 femtomoles per microliter. Include a no insert control by using one microliter of water instead of the guide RNA oligonucleotides. Digest the vector and ligate the inserts in one reaction using the Golden Gate protocol outlined in the text protocol.
After the initial golden gate reaction add an additional 0.5 microliters of the BSNB1 enzyme to each reaction. Continue the reaction at 55 degrees Celsius for one hour. When the golden gate reaction is complete proceed to the previously described E.coli transformation process.
In this study, single guide RNA expression vectors are successfully created using two methods. In the first method, the vector backbone is pre-digested and ligated in a series of short oligonucleotides. The second method used golden gate cloning to simultaneously digest and ligate in a single reaction.
Paired guide RNA expressing vectors, each driven by its own independent promoter are successfully created by cloning a custom PCR fragment. Successful cloning for either of these methods will result in the appearance of significantly more colonies for transformations with the appropriate insert DNA when compared to the no insert control plate. While attempting this procedure it's important to remember to include no insert controls in your cloning step.
Following this procedure, other methods like epigenome engineering can be performed in order to answer questions like what effects chromatin signatures have on gene expression.
