Psychology assignment

Abstract

The genetic entering aims to study the genetic composition of a cell and come up with a way of altering them to match the required characteristics. The lab report seeks to explain how CRISPR could be used to modify the genetic sequencing of a specific cell. The experiment will perform some steps to achieve the DNA that was for the experiment. Various material was used to conduct the test where each of the materials and equipment was used to obtain the desired results with the suitable temperature conditions. The results of the test were studied to assess whether the desired results were obtained and used in the discussion.

Running Head: GENETIC EDITING 1

GENETIC EDITING 6

Introduction

An organism DNA is responsible for the unique characteristics they portray. Scientists have come up with technologies that alter the genes in the DNA through a process called genome editing (Toro et al., 2013). The genome editing involves the removal, addition or altering genome in a particular location (Adame et al., 2016). Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is one the commonly known and the best genome editing technology because of its ease to use in gene editing (Abm, 2019). Therefore, the method is the best option for this experiment. The gene contains special protein with a wide range of functions. These proteins are what makes up the genome and specific method suitable for altering the gene targets the mentioned protein (Lino, Harper, Carney, & Timlin, 2018). Red Fluorescent Protein is an example which florescent red when excited. Puromycin sometimes is used to modify the genes through dissociation of ribosomes, but some of them resist the substances. HEK 293T cells is embryonic kidney cell used in mostly in cell research because of the growth reliability and tendency for transfection (Sansbury, Wagner, Nitzan, Tarcic, & Kmiec, 2018). This paper aims to report the findings of the experiment performed to prove the alteration of the gene using the CRISPR Cas9 method and the RFP gene of the human genome on the fund in Hek 293T.

Materials and methods

The experiment was performed in steps where each step aimed at producing results used in the next steps. In the bacterial transformation we thaw on ice one tube of DH5αTM cells. Place 1.5 ml microcentrifuge tubes on wet ice. Then, gently mix cells with the pipette tip and aliquot 50 μl of cells for each transformation into a 1.5 ml microcentrifuge tube. Then, refreeze any unused cells in the dry ice/ethanol bath for 5 minutes before returning to the -80°C freezer. Then, we make sure to not use liquid nitrogen. We Add 1 to 5 μl (1-10 ng) of DNA to the cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 2.5 μl (250 pg) of DNA to the cells and mix gently. Then, we incubate tubes on ice for 30 minutes. We heat the shock cells for 20 seconds in a 42°C water bath without shaking. Then, we Place tubes on ice for 2 minutes. We add 950 μl of pre-warmed medium of choice to each tube. Then, incubate tubes at 37°C for 1 hour at 225 rpm. Then, spread 20μl to 200μl from each transformation on pre-warmed selective plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, plate 100 μl on an LB plate containing 100 μg/ml ampicillin. Store the remaining transformation reaction at +4°C. Additional cells may be plated out the next day, if desired. Finally, incubate plates overnight at 37°C.

In the mini prep and Nanodrop we transfer 20-50mL overnight culture to 50mL centrifuge tube. Then, centrifuge at 4,000 x g for 10 minutes at room temperature. Then, decant or aspirate and discard the culture media. Then, add 2.25mL Solution I/RNase A. Vortex or pipet up and down to completely resuspend the cells. Then, add 2.25mL Solution II. Invert and rotate the tube gently 8-10 times to obtain a cleared lysate. This may require 2-3 minutes incubation at room temperature with occasional mixing. Then, add 3.2 mL Solution III. Invert and rotate the tube gently until flocculent white precipitates form. This may require a 2-3-minute incubation at room temperature with occasional mixing. Then, centrifuge at 15,000 x g for 20 minutes at room temperature (preferably at 4 degrees Celsius). A compact white pellet will form. Promptly proceed to the next step. Then, insert a HiBind Midi Column into mL Collection Tube. Then, transfer 5 mL cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Midi Column. Be careful bot to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Midi Column. Then, centrifuge at 4,000 x g for 3 minutes. Then, discard the filtrate and reuse the collection tube. Then, repeat Steps 10-12 until all of the cleared supernatant has been transferred to the HiBind DNA Midi Column. Then, add 3 mL HBC Buffer. Then, centrifuge at 4,000 x g for 3 minutes. Then, discard the filtrate and reuse the collection tube. Then, add 3.5 mL DNA Wash Buffer. Then, centrifuge at 4,000 x g for 3 minutes. Then, discard the filtrate and reuse the collection tube. Then, repeat Steps 17-19 for a second DNA Wash Buffer wash step. Then, centrifuge the empty HiBind DNA Midi Column at 4,000 x g for 10 minutes to dry the column matrix. Then, transfer the HiBind DNA Midi Column matrix before elution. Residual ethanol may interfere with downstream applications. Then, transfer the HiBind DNA Midi Column to a nuclease-free 15 mL centrifuge tube. Then, add 500 mL Elution Buffer or sterile deionized water directly to the center of the column matrix. Then, let it sit at room temperature for 3 minutes. Then, centrifuge at 4,000 x g for 5 minutes. Finally, store DNA at -20 degrees Celsius.

For transfection protocol we adherent Cells: 18 to 24 hours prior to transfection, seed cells at a density of 1-3 x 105 cells per well in 2.0 ml of appropriate growth medium (with serum and antibiotics if cells are cultured in the presence of them). Incubate the cells at 37 ̊C in a CO2 incubator until cells are 70% to 90% confluent at the time of transfection. Then, for each transfection sample, prepare the complexes as follows: Solution A: Dilute 2.0 μg of DNA into 100 μl of serum-free, antibiotic-free medium. Solution B: Vortex DNAfectinTM2100 thoroughly prior use, then dilute 10-20 μl of DNAfectinTM2100 in 100 μl serum-free, antibiotic-free medium. Then, incubate Solution A and B at room temperature for 5 minutes. Then, we Combine the solutions, mix gently to ensure uniform distribution and incubate for 20 minutes at room temperature. For suspension cells, go directly to step 5. We adherent Cells ONLY: Add 0.8ml of serum-free, antibiotic-free medium to DNAfectinTM 2100-DNA complex. Mix solution gently. Then, we adherent Cells: Remove growth medium from the cells and add 1.0ml of DNAfectinTM 2100-DNA solution to the each well containing cells. After 5-8 hours, remove transfection solution and add 2.0 ml of the appropriate growth medium (with serum and antibiotics) or add 0.1 ml of FBS directly into each vessel. Incubate the cells at 37 ̊C in a CO2 incubator for a total of 18-24 hours. To make stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours post transfection. Selection medium can be added the following day if desired.

In DNA Extraction we First, collect cells by scraping. Second, pellet at 8,000 x g for 10 minutes, then, wash once with PBS. Third, suspend cell pellet in TE buffer with 0.1% triton x 100. Fourth, boil at 100 degrees Celsius for 5 minutes. Fifth, centrifuge at 13,000 x g for 10 minutes. Six, collect supernatant “DNA” and store at -20 degrees Celsius till use. Last but not least, dump media off. Then, wash 1 x with mL of PbS. After that, add 1 mL PBS “leave on.” Next, scrape cells and then pipette sup cells into microcentrifuge tube.

In the Gel Electrophoresis we first, add .5g agarose in 50 mc to make 1%. Next, heat 50mL TAE buffer in microwave. Third, once gel starts to cool, add 5 mL of syber safe. Fourth, immediately pour into mold with comb. Fifth, after the gel hardens remove comb and pour TAE buffer overtop. Finally, run at 100 v for 1 hour or until labeling buffer makes it 3/<1 of the way down.

In Phalloidin Staining we first, wash cells twice with 1mL prewarmed phosphate-buffered saline, pH 7.4 (PBS). Second, in the HOOD! Fix the sample in 3.7% formaldehyde solution in PBS for 10 minutes at room temperature. Third, wash two or more times with PBS. Fourth, permeabilized the cell membranes with 0.1% Triton X-100 in PBS for 3 to 5 minutes. Fifth, wash two or more times with PBS. Sixth, when staining with any of the fluorescent phallotoxins, dilute 5 µL methanolic stock solution into 200 µL PBS for each sample to be stained. To reduce nonspecific background staining with these conjugates, add 1% bovine serum albumin (BSA) to the staining solution When staining with biotin-XX phalloidin (B7474), dilute 10 µL of the methanolic stock solution into 200 µL PBS for each sample to be stained. Seventh, place 200µL staining solution on the Mattek dish for 20 minutes at room temperature (generally, any temperature between 4°C and 37°C is suitable). To avoid evaporation, keep the lid on during the incubation. Eight, wash two or more times with PBS.

In the Lysis of Cell we first, add 250 mL of lysis buffer with protease inhibitors. Second, scrape cells with lysis buffer and transfer to new tube. Third, vortex for 30 seconds every 5 minutes for 10 minutes to break up the cells. Fourth, spin for 5 minutes at 12,000 x g at 4 degrees Celsius. Fifth, transfer supernatant to a new tube. Finally, store at -80 degrees Celsius.

In the SDS PAGE and protein transfer we made stacking and resolving gel, ran it for 45 minutes at 100 volts. Then transfer for 7 minutes.

In the western Blot we first wash in 1 x TBS 1.1% tween 20 (2x 5 min each). Second, block 5% BSA in 1x TBS 1.1% tween 20:30 minutes. Third, repeat step 1. Fourth, add a antibody for 45 minutes-1 hour. Fifth, repeat step 1. Sixth, add 3 antibody for 30 minutes RT. Seventh, repeat step 1. Eight, add a 1:1 dilution of ECL 1&2. Finally, image.

Results

The results of the experiment showed how the CRISPR method could be used in gene editing. The CRISPR method is the best gene editing method as seen from the effect obtained from the experiment. The extracted DNA as different gene composition shows are compared to the original one observed before the test was conducted. The obtained results as indicated in figure 1 had some characteristic that indicates that the repair occurred throughout the experiment (Lino, Harper, Carney, & Timlin, 2018). The extracted cells in the experimented catalyzed activities for the DNA resection, replication, and correction that was required for the plasmid linearization as shown in figure 1. The difference between both genetic compositions could be seen on the figure.

The staining of the specimen is essential for the clarity for easy identification of the changes that occurred during the experiment. The Phalloidin and DAPI stains happened to indicate the area in the nucleus which shows the changes that happened to the DNA when using a high-resolution microscope as shown in figure 2.

Also, the results portrayed on the agarose gel is a method based on the gel electrophoresis used for separation of molecules in the DNA. The figure3 shows the result obtained from the experiment.

The data obtained from the results were then used to plot a graph to show the concentration of protein in the nucleus in figure 4 — also, the data collected for the RFP for both CRISPR and non CRISPR cell. From the graph, we can observe that the CRISPR resulted in the high density of the solution which indicated that the DNA protein alteration occurred more than the other non CRISPR cells (Sansbury, Wagner, Nitzan, Tarcic, & Kmiec, 2018). This a justification that the CRISPR are the best for the genetic editing due to their simplicity characteristics it has when considering gene editing. Figure 5 shows the difference in results between the two types of Cells.

Discussion

The analysis of the result of the experiment led to the justification of the possibility of gene editing using CRISPR method. This results proved the hypothesis of possible manipulation of the RPF cells present in the DNA of the Human cell. The materials used to experiment is also important in understanding the factors that lead to the changes in the genetic sequencing in human DNA. A more complex genetic engineering can thus be performed in the genetics of an organism to match the intended characteristics, but the idea has raised ethical concerns by performing on the human gene composition. CRISPR technology is the best, and it has been used in several fields to alter the genetic structure of an organism. Also, most of the research organizations have implemented the technology to perform various research activities regarding human genetics.

References

Abm, T. (2019). CRISPR Cas9 - Tools for Genomic Activation and Repression Inc. Retrieved from https://www.abmgood.com/marketing/knowledge_base/CRISPR-Cas9-dCas9-Gene-Regulation.php

Adame, V., Chapapas, H., Cisneros, M., Deaton, C., Deichmann, S., Gadek, C., … Cripps, R. M. (2016). An undergraduate laboratory class using CRISPR/Cas9 technology to mutate drosophila genes. Biochemistry and Molecular Biology Education, 44(3), 263-275. doi:10.1002/bmb.20950

Lino, C. A., Harper, J. C., Carney, J. P., & Timlin, J. A. (2018). Delivering CRISPR: a review of the challenges and approaches. Drug Delivery, 25(1), 1234-1257. doi:10.1080/10717544.2018.1474964

Sansbury, B. M., Wagner, A. M., Nitzan, E., Tarcic, G., & Kmiec, E. B. (2018). CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract. The CRISPR Journal, 1(2), 191-202. doi:10.1089/crispr.2018.0006

Toro, M., Cao, G., Ju, W., Allard, M., Barrangou, R., Zhao, S., … Meng, J. (2013). Association of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Elements with Specific Serotypes and Virulence Potential of Shiga Toxin-Producing Escherichia coli. Applied and Environmental Microbiology, 80(4), 1411-1420. doi:10.1128/aem.03018-13

Appendix

RFP Palloidin DAPI

Figure 1 RFP, Phalloidin, and DAPI stain image. The above figure shows the RFP, phalloidin, and DAPI plasmid, this is the treated version.

Figure 2 untreated rfp Phalloidin and DAPI stain image. This figure is showing the untreated version of the RFP, Phalloidin, and DAPI

Figure 3 genome gel image.

Figure 4 Guide 1 image.

Figure 5 repair 1 image.

Figure 6 standard curve graph image.

Figure 7 SDS PAGE image.