פרוטוקול אוניברסלי עבור gRNA בקנה מידה גדול ספריית הפקה מכל מקור ה-DNA

The overall goal of this method is to construct comprehensive guide RNA libraries from purified DNA from any source. gRNA libraries are plasmid collections used in CRISPR-based screens. The CORALINA method uses enzymatic digestion of purified DNA to clone the resulting fragments into a guide RNA library backbone.

The main advantage of this technique is that it produces all possible guide RNAs from the input sequence and it is very cost-effective. Although this video demonstrates the protection of a CORALINA library from a bacterial artificial chromosome, this method can in principle be applied to any type of input DNA from any organism. An optimization of micrococcal nuclease digestion must be performed for each new batch of micrococcal nuclease.

Set up the following for each reaction. One microliter of 10x micrococcal nuclease buffer, 1x bovine serum albumin, one microgram of target DNA, one microliter of micrococcal nuclease and water to 10 microliters. Incubate at 37 degrees Celsius for 15 minutes.

Immediately inactivate the enzyme by adding one microliter of 500 millimolar EGTA. Add sample buffer with gel loading dye to the DNA samples and load one microgram of DNA per well in a 20%polyacrylamide gel. Load an appropriate DNA ladder for sizing.

Run the gel in 1x TBE running buffer at 150 volts for around 1.5 hours or until the lower dye front reaches the lower end of the gel. Stain the gel using an ultrasensitive nucleic acid stain and visualize under UV light. In this example, the optimum concentration of micrococcal nuclease for digesting the DNA down to 20 to 30 base pairs in size was 10 units.

Use this optimum concentration of micrococcal nuclease to digest 10 to 12 micrograms of target DNA in the same way as demonstrated for the optimization reactions. After separating the DNA fragments by PAGE, use a sterile scalpel to cut the gel next to the marker lane and stain only the part of the gel containing the ladder with fresh 1x TBE running buffer containing an ultrasensitive nucleic acid stain. Visualize the DNA ladder and use a razor blade to excise micrococcal nuclease digested DNA fragments in the size range between approximately 18 to 30 base pairs.

Transfer the gel slice to a microcentrifuge tube. Stain the remainder of the gel with nucleic acid stain as shown earlier. Expose the gel to UV light and acquire an image as a record of the gel excision step.

Begin this procedure by using a sterile pipette tip to crush the excised gel slice against the wall of the microcentrifuge tube. Add two gel volumes of PAGE solubilization buffer and incubate at 37 degrees Celsius on a rotating platform for 16 hours. On the following day, centrifuge the sample in a microcentrifuge at maximum speed for one minute.

Transfer the supernatants to a new microcentrifuge tube taking care not to transfer any crushed gel pieces. Add 0.5 volumes of PAGE solubilization buffer to the gel pellet, vortex and repeat the centrifugation. Combine the supernatants and extract the DNA fragments using a standard phenol-chloroform extraction method as described in the text protocol.

In parallel with the isolation of DNA fragments, amplify linkers from the guide RNA expression vector using standard PCR. To ensure directional cloning, the linkers are then digested with the appropriate restriction enzymes as described in the text protocol. Run the restriction enzyme digest of the linkers on a one percent agarose gel.

Excise the correct bands and purify the DNA from the excised gel pieces using a gel extraction kit. A DNA blunting kit is used for the end repair reaction. Add the following into a microfuge tube.

10 microliters of purified DNA, 1.5 microliters of 10x blunting buffer, 1.5 microliters of one millimolar dNTP mix, 1.4 microliters of water and 0.6 microliters of blunting enzyme. Incubate at 22 degrees Celsius for 30 minutes and then heat and activate the enzyme by incubation at 70 degrees Celsius for 10 minutes. Add 85 microliters of water and perform a reaction cleanup as described in the text protocol.

Set up a 14 microliter ligation reaction using equal molar amounts of micrococcal nuclease digested and repaired fragments and linker sequences as described in the text protocol. Following nick translation, the ligation product is amplified by PCR. To separate micrococcal nuclease fragments with the correctly attached five prime and three prime linker from fragments with two five prime or two three prime linkers, combine all 15 cycle PCR reactions, add DNA loading dye and run on a 0.8%agarose gel.

Excise the prominent band at 869 base pairs and purify DNA using a gel extraction kit. Quantify the amount of DNA using a spectrophotometer. Subsequently, clone the PCR amplified fragments into the guide RNA expression vector by Gibson assembly as detailed in the text protocol.

To begin this procedure, aliquot purified vector insert construct into sterile and pre-chilled PCR tubes and keep on ice. Chill one millimeter gap electroporation cuvettes on ice. Add 25 microliters of freshly prepared TG1 cells directly to one aliquot of DNA and immediately transfer the mixture into an electroporation cuvette.

Flick or tap the cuvette to ensure the cells and DNA mix is distributed along the length of the cuvette chamber without any trapped air or bubbles. Place the cuvette in the slide chamber and start the appropriate electroporation program. Immediately add 475 microliters of room temperature 2TY to the cuvette.

Transfer the electroporated bacteria to a 50 milliliter tube. Repeat the individual electroporations, collect all cells transformed with one library in one 50 milliliter tube and document the total volume. To estimate the total number of colonies for the entire library, Plate a defined small amount of bacteria transformed with the library on a 10 centimeter agar plate with the appropriate antibiotic selection.

To reduce the volume of the remainder of the bacteria, centrifuge at approximately 4, 000 g for 10 minutes or until a visible palette has formed and the supernatant appears clear. Remove all but a few milliliters of the supernatant and resuspend the cells. Plate the bacteria onto 2TY agar-coated bioassay dishes containing the appropriate antibiotic selection.

Spread an appropriate volume of the pUC19 control electroporation reaction on an additional 10 centimeter agar dish with appropriate antibiotic selection. Incubate the agar plates at 37 degrees Celsius overnight. On the following day, count the number of colonies on the controlled plates to estimate colony forming unit per microgram of DNA for the bacteria as well as library complexity.

Lastly, scrape the bacteria from the plate for plasmid DNA extraction. After gel purification, 1/6 of the purified micrococcal nuclease fragments were loaded onto a 20%PAGE gel to confirm successful size selection and purification. Linker amplicons were digested with restriction enzymes to ensure linkers are ligated onto the micrococcal nuclease digested fragments in the correct orientation.

The left side of this representative gel image shows five prime and three prime linkers before and after digestion with HindIII and SacII respectively, indicating complete digestion of the linkers to the predictive 637 base pairs and 295 base pairs. The right side of the gel image documents the excision of the digested linker fragments. Successful ligation of linkers to DNA fragments was analyzed using PCR with an increased number of PCR cycles.

Controls included were a no template control using the no template control from the previous nick translation step as input and a no micrococcal nuclease fragment control. Only samples in which micrococcal nuclease fragments have been combined with linker DNA produced the expected amplicon of 869 base pairs. Once mastered, this technique can be performed in five days if it's done properly.

While attempting this procedure, it's important to maintain the complexity of the library and to perform other control reactions. After watching this video, you should now have a good understanding of how to generate a guide RNA library for CRISPR-based screens by chopping up DNA into small pieces and cloning these into the library vector backbone.