In nature, Cas9 is an immune protein against invading viruses that evolution prefers Cas9 with promiscuous specificity which can cleave viral DNA with spontaneous mutations. We built an artificial directed evolution system which prefers an optimized Cas9 variant with high specificity. Both negative and positive selections work simultaneously in the Sniper Screening System.
Cas9 variants with reduced off-target activity also maintaining on-target activity can be screened at once. Mutations were introduced on whole Cas9 sequence randomly to produce the library to be screened. And this made it possible to find mutations in unexpected positions.
Currently, many researchers in academia and industry are using Y-type Cas9 for therapeutic developments. However, the specificity of Y-type Cas9 is not optimized, which can result in off tightly packed. In humans, this means unexpected mutations in random genes, which can lead to serious side effects.
There are many Cas9 like enzymes which are being developed in therapeutics. Our technique can be used to improve specificity of other DNA and nucleids like Jipinger, Thailand, and Cas9 also such as CPF1. Making a library large enough is the key to increase the possibilities to get a variant with important function.
Be sure to get a high efficiency in the cooling step, and use highly efficient competent cells. To begin this procedure, add one nanogram of both the CCDB plasmid and the SGRNA plasmid with double mismatches into fifty microliters of thawed BW25141 GOI cells. Gently mix the cells by pipetting, and transfer them into a pre-chilled, 0.1 centimeter electroporation cuvette.
Transform the E-coli with the two plasmids via electroporation. Immediately after the electroporation is completed, add 250 microliters of SOC medium. Gently pipette the solution to mix the cells and medium, and transfer the mixture to a 1.5 milliliter microcentrifuge tube.
Recover the transformed cells and incubate them at 32 degrees Celsius, with gentle shaking for one hour. Continue preparing the Snyper screening cells as outlined in the text protocol. When ready to perform Snyper screening, transform the Snyper screening cells with 100 nanograms of the prepared Cas9 variant plasmids from each library using the elctroporation process that was previously described.
Then, transfer 250 microliters of the cells to a fresh 1.5 milliliter microcentrifuge tube. Add 250 picograms of ATC to a final concentration of 10 nanograms per milliliter. Recover both the ATC-containing and the ATC-free cells by incubating them at 32 degrees Celsius for one hour with gentle shaking.
Plate 25 microliters of the recovered ATC-free cells on an LB agar plate, containing chloramphenicol and kanamycin. Add ATC to the recovered ATC containing cells to a final concentration of 100 nanograms per milliliter on a 245 millimeter LB agar plate. Immediately plate the ATC-containing cells on an LB agar containing plate containing chloramphenicol, kanamycin, and arabinose.
Incubate all plates overnight at 32 degrees Celsius. The next day, photograph the plates. Using the open CFU software, count the number of viable colonies, making sure that the number of colonies on the non-selective plate is at least ten times larger than the diversity of the library, to cover all variants.
Pull the colonies that survived on the selective plates from all three libraries. Transfer these pooled colonies to 250 milliliters of LB medium, supplemented with chloramphenicol, and incubate overnight at 42 degrees Celsius. First, load the Cas OF finder software to pick target sites.
Select the PAM Type appropriate for the specific type of Cas9 and the target genome. Fill in the query sequences tab. Choose the mismatch number and click the submit button.
After a few seconds, the on-target and off-target sites will appear. In general, choose the off-target sites with one to three mismatches. Next, order the CRRNA and the Track RRNA oligos with template sequences and amplify the template as outlined in the text protocol.
Analyze two microliters of the amplified template DNA, on a 2%agarose gel and then purify the template. Incubate the reaction mixture overnight at 37 degrees Celsius. The next day, add 0.5 microliters of DNAse and incubate at 37 degrees Celsius for 15 to 30 minutes, and then purify the SGRNA.
Then, treat 10 micrograms of the in-vitro transcribed RNA with 250 units of calf intestinal alkaline phosphatase for three hours at 3 degrees Celsius, in the presence of 100 units of RNAse inhibitor, and then purify the treated SGRNA. First, maintain HEK293 T cells in DMEM supplemented with 10%FBS and 1%antibiotics at 37 degrees Celsius with 5%carbon dioxide. Next, mix two micrograms of either wild type or Snyper Cas9 protein with two micrograms of SGRNA, and incubate at room temperature for ten minutes to make RNP complexes.
Trypsinize and count the cells, then wash them and prepare them in electroporation buffer as outlined in the text protocol. Electroporate the RNP complexes into the cells using one pulse of 1300 volts for 30 milliseconds. Immediately after the electroporation, plate the cells on a 48-well plate filled with 500 microliters of DMEM, supplemented with 10%FBS and 1%antibiotics.
Incubate at 37 degrees Celsius with 5%carbon dioxide. Maintain HEK293 T cells in DMEM supplemented with 10%FBS and 1%antibiotics at 37 degrees Celsius with 5%carbon dioxide. The day before transfection, trypsinize and count the cells.
If working at a 48-well scale, plate 10, 000 cells per well and 250 microliters of complete growth medium. Using a lipid-based transfection reagent, prepare 250 nanograms of P3S Cas9 plasmid and 250 nanograms of SGRNA for transfection. Then, mix the plasmids in 25 microliters of serum-free MEM.
Dilute one microliter of transfection reagent with 25 microliters of serum-free MEM. Incubate this mixture at room temperature for five minutes. Combine the plasmid mixture with the transfection reagent mixture and incubate at room for 20 minutes to form plasmid lipofectamine complexes.
After this, add 50 microliters of this mixture directly to each well containing cells. Gently rock the plate back and forth to mix. Incubate the cells at 37 degrees Celsius in a carbon dioxide incubator for 48 to 72 hours post-transfection, before assaying for trans-gene expression.
After isolating the previously prepared genomic DNA, generate deep sequencing libraries by PCR amplification of the GDNA with primers targeting on-target and off-target. Then, use index primers to label each sample. Use a next-generation sequencing machine to subject the pool libraries to paired end sequencing.
After the Snyper screen is performed, the percentage of survival colonies can be calculated by dividing the number of colonies on the selective LB plate by the number of colonies on the non-selective LB plate. While this percentage is usually very low when performed with libraries of SP Cas9, true positive hits can be enriched by repeating the screen with the surviving pool. A representative Snyper screen shows a 100%survival rate is obtained after the third screen.
Transfections using RNPs or plasmid encoded Snyper Cas9 can be done for various targets, and the resulting on-target and off-target activities measured by targets amplicon sequencing. At most targets, Snyper Cas9 shows the same level of on target activities and higher specificity ratios compared to the wild type. Truncated SGRNAs can also be used to further improve specificity.
Higher transformation efficiency in the first cleaning step is very important not to lose the clones with high specificity. Later screening steps recur less transformation efficiency since the clones are already enriched in the first screening step, and there is less chance of losing them. There will be more than one clone that we found in Snyper screen.
On-target and off-target activities of these clones should be characterized in as many different targets as possible to select clones with the best performance. With this method, scientists can improve the specificity of the Cas9-like enzymes without loss of on-target activity. In addition, scientists do not need any prior knowledge on the structure of the protein to make improvements.
