This method can help answer key questions in the field of genomics and gene regulation, such as how multiple gene regulatory regions, also known as enhancers, work together to control transcription. The main advantage of this technique is that multiple enhancers near multiple different genes can be tested simultaneously in order to rapidly identify regions that are involved in gene regulation. To begin guide RNA design, first generate DNA sequences as described in the text protocol.
Use a program such E-CRISP on the generated DNA sequences to find guide RNAs with low off-targets. Guide RNAs consist of 20 nucleotides upstream of a protospacer-adjacent motif, which takes the form of NGG for the dCas9 from S.pyogenes. On the E-CRISP website, select the organism of interest using the drop-down menu.
The genome assembly appears to the right of the species name. Select the Input is FASTA sequence radio button. Copy the FASTA sequences from above and paste them into the dialog box.
Ensure that a FASTA header is included for each sequence. Select the medium radio button and Single design in the drop-down menu. Click the button Start single gRNA search.
A new browser tab will open and results will be displayed. Download the candidate sequences by clicking the button, Download an Excel formulated tabular report for all query sequences together. Next, use the UCSC genome browser to BLAT candidate full length guide RNA sequences to the genome.
In a browser, navigate to the UCSC genome browser website. Under the section Our Tools, locate the word BLAT and click on it. The BLAT search tool will open.
Use the dropdown menus located under the BLAT search genome text to select the organism and genome assembly of interest. Copy the guide RNA sequences from the tabular report generated by E-CRISP and paste them into the dialog box. Ensure that each sequence has unique FASTA header, then click submit at the bottom of the dialog box.
On the BLAT search results page, alignments of each guide RNA sequence will appear, with each line representing an alignment. Ideally, there should be one alignment for each guide RNA, indicating the uniqueness of that guide RNA. Avoid guides that map to multiple locations in the genome, if possible.
To examine guide RNA localization and distribution within the region of interest, click on the browser link under the Actions section for one of the queried guide RNAs. The genome browser will appear, and will be centered on the selected guide RNA. Use the zoom out buttons at the top of the page to visualize the distribution of other guide RNAs identified by E-CRISP within the region of interest.
Select four, preferably non-overlapping, guide RNAs that are distributed throughout the region of interest. If the region of interest exceeds 600 base pairs, consider adding one to two additional guides. Avoid guide RNAs with homopolymeric stretches and extreme GC content, as these features can hinder the guide RNA cloning process and reduce guide RNA targeting efficiency.
Reconstitute guide RNA oligos at a final concentration of 100 micromolar in ultra-pure water. There should be at least four separate guide RNA oligos for each region of interest. For each regulatory region of interest, create a pool of all the oligos corresponding to the region of interest.
In an Eppendorf tube, combine five microliters of each individual reconstituted guide RNA oligo for each region. Mix the pool well by vortexing, then remove one microliter and dilute this eloquat 1-to-200 in ultra-pure water. Perform a short PCR with the USICS primers to attach homology regions to the oligos as detailed in the text protocol.
About 40 bases will be added to each oligo, yielding an approximate 100 base pair product that contains sufficient homology to the USIC specter on both ends. Next, set up Gibson Assembly reactions on ice. Use 50 nanograms of the digested vector and seven nanograms of the insert in a 20 microliter reaction.
Also, set up a digested-vector-only Gibson Assembly reaction using 50 nanograms of the vector, and replacing the insert with water. Incubate the Gibson Assembly reactions for 15 minutes at 50 degrees Celsius, followed by a hold at 4 degrees Celsius. After transferring the assembled products to ice, dilute them 1-to-4 in ultra-pure water on ice.
For example, add five microliters of Gibson Assembly product to 15 microliters of ultra-pure water. Next, transform the diluted Gibson Assembly products. Fall high-efficiency competent cells on ice, and make 25 microliter eloquats for each transformation.
If a complex pool targeting multiple sites is desired, fall enough cells into different tubes to perform multiple independent transformations. Plate 50 microliters of the transformed cells, and place the plates in a 37 degrees Celsius incubator overnight. For mini-preps of the pools targeting individual sites, place the cells directly in three to five milliliters of LB broth containing ampicillin or carbenicillin.
Incubate the cells overnight with shaking at 250 rpm, and 37 degrees Celsius. For large libraries, use a plate scraper to collect all the colonies from each individual plate into one maxi-prep. This can be facilitated by pouring approximately five milliliters of LB with the appropriate antibiotic into a 15 milliliter Falcon tube, and scraping the colonies into the tube.
The day before transfection, plate the cells in a 24-well plate, at 30 to 50%confluency. Plate enough cells such that transfections can be performed in duplicate, and include wells to be transfected with control guide RNAs. Ensure that the cells are evenly distributed across the well, by gently shaking the plate after cell plating.
Shake every five minutes in the first 15 minutes. The following day, prepare transfections as per instructions of the transfection reagent of choice. For Ishikawa cells, use 550 nanograms of total plasmid for each well of a 24-well plate.
Dilute the plasmids to a final concentration of 020 micrograms per milliliter in serum-free media. Use three microliters of transfection reagent for every one microgram of DNA, vortex gently, and incubate for five to 10 minutes at room temperature. Add 25 microliters of the final mixture to each well.
Prepare a sufficient volume of lysis buffer with 1%beta-mercaptoethanol. Aspirate the media using a vacuum aspirator. Wash the cells once with an equal volume of 1x PBS, and aspirate to remove as much PBS as possible.
Add 300 microliters of the lysis BME solution to each well, using a multi-channel pipette. Pipette the lysis solution up and down eight to ten times, and transfer to a deep well plate or 1.7 milliliter Eppendorf tubes on ice. RNA can be extracted immediately, or lysates can be frozen at minus 80 degrees Celsius for future processing.
Guide RNA designs are shown for the three transcription factor binding sites near MMP17. The target region is the binding site for ER alpha as defined by ChiP-seq, and the four guide RNAs tile across this region. Shown here is the expected guide RNA product after a short PCR, using the USIC's internal primers.
The reaction will add 20 base pairs of sequence to each end of the 59 base pair guide RNA fragment, resulting in an approximate 100 base pair sequence. Qualitative PCR results from an Enhancer-I experiment at MMP17 are shown. Sites targeted by Enhancer-I are indicated with a black hexagon.
Sites 1 and 2 are necessary for complete estrogenic response of MMP17, while site 3 does not contribute. Site 1 can contribute some expression by itself, but the greatest activity is seen when sites 1 and 2 are active. Once optimized for the cell line of interest, this technique can be done in five to seven days, if it is performed properly.
After watching this video, you should have a good understanding of how to design guide RNAs for Enhancer-I, create and transfect complex pools of guide RNAs, and harvest transfected cells. While attempting this procedure, it is important to maintain a sterile tissue culture environment, and use RNase-and DNase-free materials. Following this procedure, other methods, like ChiP-seq and RNA-seq, can be used to answer additional questions, such as where in the genome Enhancer-I is binding, and how it is affecting global gene expression.
After its development, this technique paved the way for researchers to explore estrogen-induced gene regulation at endogenous loci in the human genome, instead of using episomal reporters. Don't forget that working in mammalian tissue culture settings can be hazardous, and precautions, such as wearing personal protective equipment, should always be taken while performing this procedure.
