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Summer Research Fellowship Programme of India's Science Academies

Genome editing by delivering CRISPR/Cas9 in glioma cells and checking mutation

Yashasvi Sharma

Lachoo memorial college of science and Technoloy, Jodhpur 342001, Rajasthan

Dr. Manzoor Koyakutty

Amrita centre for Nanosciences and Molecular Medicine, Cochin 682041

Abstract

Clustered Regularly Interspaced Palindromic Sequence (CRISPR) is a gene editing technology causing a major breakthrough in biomedical research. It brings the possibility of knock in/out of genes from cell and to correct errors from genome. Because of its quick, cheap and ease in procedure it is versatily used. Cas9 is an RNA guided DNA endonuclease enzyme associated with CRISPR which recognises specific location of the target DNA where the cut has to be made presented by guide RNA sequence after entering in cell, during S phase. Cas9 sequence complementary to gRNA which help to induce site directed double strand cut in the target location of genome. It is present in bacteria as acquired immune response against viruses. T98 cell were treated with CRISPR Cas9 for targeting MGMT gene for exon region 3 by transfection agent lipofectamine (Invitrogen, USA) which was kept for incubation for 72 hours. The presence of Cas9 and its stability was confirmed by performing western blot. The cell lysate was taken and T7 endonuclease assay was performed to detect CAS9 induced mutation in cells. The sample was subjected to agarose gel electophorosis having cells with and without Cas9 enzyme along with DNA ladder. Sample was purified from any dimers or tetramers using PCR gel purification kit. Hybridization was performed using resolvase enzyme to check whether Cas9 was capable to cleave the targeted location of exon region 3 on the MGMT gene. After completion other techniques like immunoflouroscence staining of RAW 264.7 macrophage cell line derived from mouse was done for actin and nucleus were spotted using DAPI and FITC dyes. We also learned isolation of monocytes and lymphocytes from human blood using peripheral blood mononuclear cell isolation (PBMC) protocol.

Keywords: CRISPR, Cas9, gRNA, Western Blot, T98 cells, MGMT

Abbreviations

Abbreviations
CRISPR Clustered Regulate interspaced paliindromic repeats 
gRNAGuide Ribonucleic acid 
                                   CASCRISPR associated sequences 
AGE  Agarose gel electrophoresis
 PBMC peripheral blood mononuclear cells
 WB western blot
 DNA Deoxyribonucleic acid
 ATCC American type cell culture
PVDF  Polyvinylidene fluoride
 HRPhorse radish peroxidase 
 ul micro litre
KDa  Kilo Dalton
 mlmilli litre 
APS 

 ammonium persulphate
 TEMED Tetramethylethylenediamine
 SDSsodium dodecyl sulphate 
 HCl Hydrocholric acid
TGS  Tris Glycine SDS
mM milli molar 
 gmgram 
M  Molar
TBS tert-Butyldimethylsilyl chloride 
 RPMrevolution per minute 
 PMSFphenylmethylsulfonyl fluoride 
 DMSODimethyl sulfoxide 
 ngnano gram 
 mgmilli gram 
 RBC Red blood cells

INTRODUCTION

Background

CRISPR/Cas9 system is a powerful bacterial defence mechanism system against viral and phage infection. It is a powerful RNA guided DNA targeting platform for genome editing, transcriptional perturbation, and genome imaging. It enables the researcher to precicely manipulate virtually any segment of genome using short pre designed sequences of RNA, the guide RNA. This helps to unravel the gene function in disease development, progresssion and treatment along with correction of disease causing mutation, knock out of oncogenes, or suppression/activation of genes. Ideally the gRNA sequence is having homology with the genomic DNA. Cas9 will only cleave the segment when the gRNA spacer sequence is present.once the cas 9 gRNA sequence binds to the target location 8-10 bases of the gRNA will begin to anneal the targeted DNA. It will only happen between the homologus sections of DNA and gRNA, at non-complementary segment breaking is abolished.

crispr-cas9_process_yourgenome_1.png
    Mechanism ofCas9 gRNA action 

    Commercially available transfection agent lipofectamine CRISPRMAX is avaliable which helps in transport of Cas9 gRNA complex across the negatively charged Plasma Membrane. The gRNA Cas9 complex is negatively charged so it cannot be directly send to the cytoplasm. Rather a ransport vehicle is required which can overcome this barrier, here we used Lipofectamine CRISPRMAX Cas9 transfection agent by Thermo Fischer. It is lipid based agent highly effectient delivery agent of Cas9 gRNA complex. The complex is coated by the lipid which is similar to the phospholipids present in the cell membrane forming a cationic liposome. Then this lipid encapsulated complex can pass the cell membrane and reach the cytoplasm.

    cationic-lipid-mediated-transfection-mechanism.jpg
      mechanism of Lipofectamine  https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/transfection-basics/gene-delivery-technologies/cationic-lipid-mediated-delivery/how-cationic-lipid-mediated-transfection-works.html

      T7 Endonuclease I (T7E1) detects on-target CRISPR/Cas9 editing events in cells. In this method, a sample of the edited cell population is used as a direct PCR template for amplification with primers specific to the targeted region. The PCR product is then denatured and reannealed to produce heteroduplex mismatches where double-strand breaks have occurred, resulting in insertion/deletion (indel) introduction. These mismatches are recognized and cleaved by T7Endonuclease and the cleavage is easily detectable and quantifiable by gel analysis. CRISPR introduced double-stranded breaks (DSBs) at desired target sites can be repaired by nonhomologous end joining (NHEJ), which is error prone and typically results in small insertions or deletion. The target region is PCR-amplified, and the PCR products are denatured and re-annealed to allow mismatched DNA to form. T7 endonuclease can recognize and cleave such mismatched DNA. By incubating the re- annealed PCR fragments with T7 endonuclease, if two shorter bands of the predicted size are generated, it usually means that CRISPR has successfully introduced mutations at the targeted chromosomal site.(Adriano Flora, PhD&Jochen Welcker, PhD)

      E3321Mutation_Detection_workflow-300x282.jpg
        Working of T7 endonuclease assay.

        The guide RNA is supplemented with the location of R3 exon region on the MGMT gene. Sucessful transport and stabiliy of Cas9 gRNA is confirmed by western blot after incubation of 72 hours. From the cell lysate the recquired segment connsisting of R3 fragment is amplified using PCR which is then subjected to AGE for sepration of the desired fragment. The isolated band is purified using gel purification kit. To the isolated purified gel Resolvase enzyme is added for the crucial step of hybridisation. Using AGE the sample is checked for 2 bands under UV trans illuminator.

        t98_control._10x.jpg
          T98G cells as veiwed under bright feild microscopy

          Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay for detection and characterization of proteins. It is based on the principle of immunochromatography where proteins are separated into polyacrylamide gel according to their molecular weight. The protein thus separated are then electrotransferred onto PVDF membrane and are detected using specific primary antibody and secondary enzyme labeled antibody and HRP substrate

          PCR allows a specifically targeted DNA sequence to be copied and/or modified in predetermined ways. This reaction has the potential to amplify one DNA molecule to become over 1 billion molecules. This powerful and versatile technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA (site-directed mutagenesis). PCR is also useful for determining whether a particular DNA fragment is found in a cDNA library. Today, PCR has been further developed to include many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and quantitative PCR which allows for quantitative measurement of DNA or RNA molecules.

          Agarose Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix towards a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. Thus, we can determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder which is a collection of DNA fragments of known lengths.

          Objectives of the Research

          To deliver Cas9 gRNA complex by CRISPRMAX in cell lysate prepared from T98G cell from ATCC source - Human and studying its stability by western blot and check mutation has been caused in the R3 region exon of MGMT gene by T7 endonuclease assay.

          LITERATURE REVIEW

          The novel technique of genome editing by CRISPR is spectacular and promising discoveries made in biology. Derived from the natural source, bacteria who uses it agains viruses and bacteriophages. When the first encounter between virus and bacteria occurs, the latter captures small segment of DNA of attacking virus and use them to create DNA sequence called as "CRISPR array". This CRISPR array allows bacteria to remember virus and use them against virus upon subsequent encounters, short DNA sequences called as protospacers adjacent motifs (PAM) tells the target location to Cas9. Its advancement lies in the fact that it can cleave both the strands of DNA.

          The system consist of:

          • Cas9 enzyme- molecular scissor which make cut at the specific location of the genome. It is transported along with lipofectamine CRISPRMAX.
          • gRNA:20 base pair pre designed RNA sequence present in larger RNA scaffold. The larger part binds to DNA whereas the gRNA guides Cas9 where to make cut. Its bases should be complementary to the target genomic DNA.

          CRISPR offers several advantages

          • In treatment of cancer, Hepatitis B, and even lowering the cholestrol.
          • It is also helpful in editing the germline cells so that the characters can be knocked in or out of the genotype in future generations.
          • Arguably, the most important advantages of CRISPR/Cas9 over other genome editing technologies is its simplicity and efficiency.
          • It does not require any cleaving enzyme from the outside, hence making it self sufficient.
          • The Cas9 enzyme can literally be matched with any gRNA sequence. It evicts the need of complementary matching for best results.
          • It can target multiple gene locations.
          • It has proven useful in repairing defective DNA in mice.

          The future aspects includes its current studies being followed on animal models to be soon avaliable for human uses. It also has to be focussed on elimination of off target impact on the genome. Critical analysis over this may leads to a method development where all the favourable and useful locations of the genome can be grouped together.

          Limitations of CRISPR

          • The effect of off-target can alter the function of a gene and may result in genomic instability, hindering it prospective and application in clinical procedure.
          • CRISPR/Cas9 still is not 100% precision, which means that still off-targets occur upon engineering the genome and this can result in undesired aberrant phenotype.

          METHODOLOGY

          Aim: To prepare cell lysate from T98G cells present in suspension.

          Materials Recquired:

          • T98 cells grown in suspension
          • Ice cold Cell Lysis Buffer
          • Phosphatase Inhibitor Single-Use Cocktail (100X) (Thermo Scientific #78442)
          • Ice cold PBS
          • Ice
          • Centrifuge tubes
          • Micropipettes and tips

          Protocol for cell lysate preparation

          1. Culture cells to a density of 1-2 million cells/ml.

          2. Washcells 3 times with 5 ml ice cold PBS.

          3. Spin 12500 rpm for 10 minutes. Decant the PBS wash and aspirate the excess supernatant.

          4. To 2ml cenrifuge tube add 2ml cell extraction buffer along with 100ul of Protein Inhibitor Complex(PIC) and 7ul of PMSF (0.3M PMSF in 50ul DMSO).Vortex this mixture.

          5. Take 200ul of the above solution into centiguge tube which contains the pellet.

          6. Incubate in ice at 4 degree celsius for 30 minutes. vortex every 10 mins for 3 times.

          7. Centrifuge at 13000 rpm at 4 degree for 10 mins.

          8. Store the supernatant at -80 degree celsius.

          Aim: To confirm the presence of CRISPR Cas9 transfected by lipofectamine inside cell after 72 hours bywestern blot

          Materials recquired:

          • Micropipettes and tips
          • 1.5mm plates(Biorad)
          • Gel Caster
          • 20 ml beakers
          • Fume hood
          • 1.5 mm 10 well plate(Biorad)
          • Centifuge
          • Dry bath(95 degree celsius)
          • Gel transfer apparatus
          • Shaker
          • Sponge
          • Filter paper
          • PVDF membrane
          • Forcep
          • Gel roller
          • electrophoresis chamber system
          • Membrane transfer unit

          Reagents Required

          sample loading reagents and concentration
           T98 cell treated withCas9 20ul
          U87 variant III as control 20ul 
           Leamlii buffer 20ul
           DNA Ladder 250 KDa 2ul
          composition of resolving gel(10%, 10ml)
           Acrylamide3.3ml 
           1.5MTris HCl pH8.8 2.5ml
           10%SDS 100ul
           milliQ water 3.96ml
           10%APS 100ul
           TEMED 10ul
          Note
          add the gel into gel caster and leave for 20 minutes. Add isopropanol at top to level the gel.
          Composition of stacking gel (4%, 5ml)
           Acrylamide 650ul
          Stacking buffer (0.5M tris pH6.8+0.4%SDS)1.25ml 
           milliQ water 3.05ml
           10%APS 25ul
           TEMED 5ul
          Note
          add stacking gel over resolving gel, place 10 well comb and leave for 2 hours
          Running Buffer(10M TGS)
           25mM Tris 30.28 gm
           190mM Glycine 144gm
           0.1%SDS 10gm
          Note
          mix all the reagents and make the volume 1 litre. To make 1Molar add 100 ml TGS and 900ml water.
          Composition of Transfer Buffer
           10M TGS 100ml
           water 700ml
           Methanol 900ml
          Note
          use this buffer cold
          washing buffer(TBST)
          TBS  10ml
           water 90ml
           tween 20 200ul
          Blocking solution
           TBST 4ml
           5% milk powder 0.2gm

          Protocol for WB

          1.Prepare control (C) loading samples (Cas9 with concentration of 20ng/ul) by diluting 20ul of control, 10ul Leamelli Buffer

          2.Prepare test (T) loading samples (U87 variant III with concentration 10ng/ul ) by diluting 10µl of T98 cell lysate, 10 ul of Leamelli Buffer

          3.Centrifuge the control and test samples at 6,000 rpm for 10 seconds

          4.Heat samples at 95 °C in a dry bath for 5 minutes.

          4.After heating the samples are again centrifuge at 6000 rpm for 10 seconds.

          5. Prepare the SDS-PAGE gel by inserting it into the electrophoresis apparatus and filling with running buffer. Remove the well plate. Rinse the wells of the gel with running buffer and verify there are no bubbles.

          6. Load the 15 ul of control and test samples in appropriate well.

          7. Load the 2ul of prestained molecular weight marker(DNA Ladder) in the first well. For monitoring the separation during electrophoresis, and subsequently variefy the protein sizes during analysis.

          8. Run the gel until the loading buffer reaches the bottom. This is typically 60-70 minutes at 150V.

          9. Remove the gel from the cassette and float in transfer buffer.

          10. Take PVDF membrane and for activation place in methanol for 2 minutes. Incubate membrane in cold transfer buffer for 10 minutes.

          11. Set up gel/ membrane sandwich by placing the transfer cassette in cold transfer buffer. Create a sandwitch stack by placing the components such as sponge, filter paper, gel, membrane, filter paper, sponge from the cathode to the anode. So that the negatively charged proteins moves from the gel into the membrane.

          12. Use a clean roller with layer to gently roll out any bubbles present.

          13. Lock the cassette and place in the transfer apparatus containing cold transfer buffer at 4 degree celsius.

          14. Perform the transfer at 100V for 60minutes at 4 degree.

          15. Remove the membrane from the cassette and wash three times inTBST.

          16. Add blocking solution and keep in shaking platform for 1 hour at room temperature.

          17. Decant the blocking solution and wash with TBS-T for 5 minutes for 3 times.

          18. Dilute the primary antibody by adding 4ml of 5%milk powder and 1.3ul of primary antibody (anti GFP) into the membrane giving 1:3000 dilution.

          19. Incubate membranes with primary antibody overnight at 4°C on with gently shaking.

          20. After incubation, decant the primary antibody and wash membrane with 5ml of TBS-T and vigorous agitation 3 times for 10 minutes each.

          21. Dilute the secondary antibody by adding 4 ml of non–fat dry milk solutionand 0.2 µl of secondary antibody (anti- mouse antibody) in to the membrane.

          22. Incubate membranes with secondary antibody for 2 hours at room temperature with gentle shaking

          23. Decant the secondary antibody and wash membrane with 5ml of TBS-T and vigorous agitation 5 times for 10 minutes.

          24. To membrane add 200 ul of HRP, use a tissue paper to wipe off excess solution from the corner of the membrane. Place the membrane in a clear plastic wrap, such as a sheet protector, to prevent drying. Do not let the membrane completely dry out.Incubate for 2-3 minutes and visualise under chemi doc imager.

          Aim: To carry out T7 endonuclease assay on MGMT gene using PCR and check for bands by AGE

          ​T7 Endonuclease recognizes and cleaves non-perfectly matched DNA.To determine genome targeting efficiency by digesting annealed PCR products with T7 Endonuclease. In the first step PCR products are produced from the genomic DNA of T98G cells whose genomes were targeted using Cas9. In the second step, the PCR products are annealed and digested with T7 Endonuclease . Fragments are analyzed to determine the efficiency of genome targeting after hybridisation with resolvase.

          Protocol for PCR

          Add all the reagents then add 2ul of sample and quick spin for 6000 RPM.Amplify the nucleic acids using the denaturation, annealing, and polymerization times and temperatures listed below.

          After completion incubate at 4 degree celsius for 10 minutes.

          Protocol for AGE

          Materials Recquired:

          1. Agarose solutions

          2. Ethidium bromide.

          3. Electrophoresis buffer

          4. DNA samples

          5. DNA Ladders.(1000 base pairs)

          6. An electrophoresis chamber and power supply.

          7. Gel casting trays

          8. Sample combs, around which molten agarose is poured to form sample wells in the gel.

          9. Electrophoresis buffer-TBE

          10. Loading buffer

          11. Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.

          12. Ethidium bromide, a fluorescent dye used for staining nucleic acids.

          Procedure

          1. Prepare 1M TBE buffer, 20ml into conical flask.

          2. Weigh 0.2grams of agarose and add to the20ml buffer solution to make it 1% agarose gel.

          3. Keep in oven for 30 seconds, at intervals take out and swirl. do not let it boil.

          4. Take the solution from oven.

          5. Add 1ul ethidium bromide.

          6. Pour the solution to a gel caster.

          7. Place the comb (1mm 10 wells).

          8. Pour the 20ml buffer solution to the electrophoretic chamber.

          9. Place the gel in the caster in the electrophoretic chamber and leave for 20-30 minutes.

          10. Remove the well plate and add samples:

          T98 cells: 5ul sample + 3ul loading buffer (untreated and trearted with lipofectamine); Ladder: 3ul ladder+ 2ul loading buffer

          11. Connect the electrodes and switch on the current.Run for 120V for 20 minutes.

          12. Switch off the power supply.

          13. Remove the gel from the electrophoretic chamber.

          14. Place the gel in the UV Transilluminator and image it.

          Aim: To purify band from the gel using QIAquick Gel Extraction Kit

          Protocol

          1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

          2. Weigh the gel slice in a colorless tube. (Control weight was 550mg and treated weight was520mg ).Add Buffer QG to the control and treated tubes with gel(1100ul and 1040ul respectively).

          3. Incubate at 50°C for 10 min until the gel slice has completely dissolved. Vortex the
          tube every 2–3 min to help dissolve gel.

          4. Place a QIAquick spin column in a provided 2 ml collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 minute.

          5. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.Centrifuge the QIAquick column in the 2 ml collection tube for 1 min to remove residual wash buffer.

          8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

          9. To elute DNA, add 50 μl Buffer NE (5 mM Tris·Cl, pH 8.5) and centrifuge the column for 1 min. let the column stand for 1 min, and then centrifuge for 1 min.

          10. Take the concentration of isolated DNA by nanodrop.

          WhatsApp Image 2019-07-24 at 1.51.55 PM.jpeg
            Reagents used in PCR gel purification
            WhatsApp Image 2019-07-24 at 1.54.45 PM.jpeg
              Left: PCR gel clean up column; Right: collection tube

              Aim: To detect mutation caused on MGMT gene Region3 exon by Lipofecamine transfected Cas9 gRNA by Guide-it Mutation Detection Kit

              1. Mix 10 µl of PCR product and 5 µl of PCR-grade water in a PCR tube.

              2. Add 15 µl of positive control DNA to PCR tube.

              3. Perform DNA hybridization as follows:

              95°C 5 min

              95°C–85°C 2°C/sec

              85°C–25°C 0.1°C/sec

              Cool to 4°C

              4. Add 1 µl of Guide-it Resolvase, and incubate at 37°C for 15 min.

              5. Run reaction on 2% agarose gel.

              6. Determine the cleavage efficiency using a gel imaging system and gel analyse the relative proportions of the bands produced.

              Observations and Results

              Several optimisations in western blot has to be made. For Cas9 No. of washes, primary and secondary antibody dilution, choice of blocking solution all has to be experimented and tried to optimise. Like:

              • Resolving gel 10%
              • Stacking gel 4%
              • 3 washes for 10 minues after addition of blocking solution, Primary antibody, and secondary antibody.
              • Dilution for Primary antibody-1:3000
              • Dilution for secondary antibody- 1:20000
              • HRP substrate concentration - 400ul
              Slide1_7.JPG
                Left:blot image showing size of DNA ladder; Right:showing Cas9 band along with ladder and control

                Following WB we did PCR and AGE for amplifying the targeted gene region on MGMT by supplementing with suitable primers, template and polymerase. R3 region band size of 1000 base pair was confirmed by DNA ladder of size 1500 Da.

                Slide1_2.JPG
                  well 1: DNA ladder; well 2&3 control: well 4: Cas9 untreated(control), well 5: Cas9 treated

                  After getting the desired band we purified the band with gel purification kit and took the concentration of DNA using nanodrop spectrophotometer.

                  WhatsApp Image 2019-07-25 at 11.31.56 AM (1).jpeg
                    listed 1 and 2 represents data of cas9 untreated; 3 and 4 is representation of Cas9 treated with lipofectamine CRISPRMAX.

                    Following gel purification we did mutatin detection assay by Guide-it Mutation Detection Kit.

                    Slide1_3.JPG
                      well 1: DNA ladder; well 2&3 control: well 4: Cas9 untreated(control), well 5: Cas9 treated

                      Other Techniques Learned

                      RNA isolation by TRIZOL Method

                      A. Required reagents:

                      1. Double autoclaved water

                      2. TRIzol Reagent (Invitrogen)

                      3. Ice cold PBS

                      4. 75% ethanol

                      5. Isopropyl alcohol

                      B. Equipment:

                      1. Refrigerated centrifuge

                      2. Micropipetts and tips (Double autoclaved)

                      3. Vortex mixer

                      a. Spin cells for 5 min at 1200 RPM . Remove media and resuspend cells in ice cold PBS 5ml. Pellet cells by spinning at 300 X g for 5 min.

                      b. Lyse cells with 400ul TRIZOL Reagent by repetitive pipetting. Incubate the homogenized sample for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.

                      c. Add 80ul of chloroform. Cap sample tubes securely.

                      d. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at 12,000 x g for 15 minutes at 20 degree celsius. Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase.

                      e. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube.

                      f. Precipitate the RNA from the aqueous phase by mixing with 200ul isopropyl alcohol at room temperaure Incubate samples at for 10 minutes and centrifuge at 12,000x g for 10 minutes at 4 degree. The RNA precipitate, forms a gel-like pellet on the side and bottom of the tube.
                      g. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, 400ul. Centrifuge at 7,500 xg for 5 minutes at 4 degree.

                      h. Air-dry or vacuum dry RNA pellet for 5-10 minutes. Resuspend in 40ul of double autoclaved water. Store at -80 degree.

                      Slide1.JPG
                        Layers of RNA and DNA obtained during TRIZOL Treatment
                        WhatsApp Image 2019-07-25 at 10.23.39 AM.jpeg
                          Nanodrop Result obtained for RNA isolation.
                          Slide1_4.JPG
                            RNA on agarose gel

                            Result: At some point RNA have been lost. So clear bands were not seen.

                            Phalloidin staining for Actin and nucleus in Raw mouse macropages 264.7 extraced from glioma cells

                            Reagents recquired:

                            • 4% PFA (Paraformaldehyde)
                            • 0.1%Triton X100
                            • 1% FBS (Foetal Bovine Serum)
                            • FITC (Fluorescein isothiocyanate)
                            • DAPI (4′,6-diamidino-2-phenylindole)
                            • Invitogen Prolonged Diamond Antifade Mount
                            • PBS (Phosphate-buffered saline)

                            STEPS

                            1. Culture Cells on coverslips inside a well plate.

                            2. Aspirate cell culture medium.

                            3. Wash once in PBS.

                            4. Fix cells in 4% formaldehyde 200ul at room temperature for 20 minutes.

                            5. Aspirate fixation solution and wash cells 3 times in PBS.

                            6. Add 0.1% Triton X-100 200ul into the fixed cells for 5 minutes to increase permeability. Then wash cells3 times in PBS.

                            7. Block with 1% FBS 100ul for 30 mins and wash with PBS 3 times.

                            8. Add 20ul phalloidin-conjugate FITC solution. Incubate at room temperature for 1 hour.

                            9. Rinse cells 2 times with PBS.

                            10. Add 20ul DAPI solution and leave for 5 minutes. Wash with PBS, 3 times.

                            11. Add mounting media (Invitogen Prolonged Diamond Antifade Mount) 1 drop.

                            12. View under fluoroscent microscope.

                            ys_1.jpg
                              Fluoroscence Microscopy

                              PBMC Isolation from human blood

                              A peripheral blood mononuclear cell (PBMC) is any peripheral blood cell having a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nuclei, and granulocytes (neutrophils, basophils, and eosinophils) have multi-lobe nuclei. Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and monocytes. PBMCs are separated from the whole blood by a density gradient centrifugation method using Ficoll Histopaque.

                              Materials and Reagents

                              1. Freshly collected heparinised blood

                              2. Ficoll Histopaque

                              3. Sterile PBS

                              4. Dulbecco's modified eagle medium

                              5. Pencillin-streptomycin solution (1%)

                              6. Heparin vials

                              7. Centrifuge machine with swing-out bucket rotor (Eppendorf 5810 R )

                              8. Sterile 15 ml centrifuge tube

                              9. Auto pipettes and 1 ml tips ​

                              Protocol

                              1. Mix 1 ml of blood with 1 ml of PBS.

                              2. Overlay the mixed blood solution on top of the Ficoll-Paque PLUS in 3:4 ratio.

                              3. Centrifuge at 400 x g for 30 minutes at room temperature at accelaration of 1 and deceleration 0.

                              4. Take 45ml Basal media, add:

                              complete media composition
                               PenicillinStreptomycin (anti anti)500ul 
                              FBS 4.5ml 
                               Beta mercaptoethanol50ul 
                               L glutamic acid500ul 
                               Na Pyruvate500ul 

                              5. Extract the second white layer carefully into a fresh tube.

                              6. Wash with 3 times the volume of Basal Media and Centrifuge at 180g for accelaration and decelaration 9.

                              7. Resuspend pellet in 1ml complete media.

                              Slide1_5.JPG
                                Layers seperation 

                                CONCLUSION

                                Cas9 gRNA complex is highly negative hence it was transported by commercially avaliable agent of lipofectamine,CRISPRMAX. After incubating cells for 72 hours cell lysate was drawn from the T98G cells the stabiity of CAS9 inside the cells were studied by performing WB where the band of Cas9 of size 160 KDa was obtained after comparing it with the path of DNA ladder of size 250 KDa. To move further we performed T7 endonuclease assay to check mutation; carried out with the help of PCR and AGE. The site of mutation was region 3 exon on the MGMT gene. The assay cleaves non-perfectly bounded segments of DNA.

                                T7 endonuclease recognizes and cleaves structural deformities in DNA heteroduplexes. Genomic DNA of a population of cells treated with CRISPR-Cas9 reagents will be amplified by PCR. If a non-homologous end joining (NHEJ) repair event following CRISPR-Cas9 cleavage has introduced a mutation, denaturing and annealing will form heteroduplexes PCR amplicons. T7 endonuclease will recognize and cleave DNA mismatches in those heteroduplexes. Running the cleavage products on a polyacrylamide or agarose gel, will resolve full length and cleavage products.

                                The agarose gel was cleaned from any polymers using PCR gel purification kit. According to nanodrop studies good yeild of DNA was obtained. Following purification, mutation detection study was done by relevent kit. 2 bands were obained after treating with resolvase proving that cut has been made successfully.

                                REFERENCES

                                Wang, T.,Wei, J.J.,Sabatini, D.M.&Lander, E.S.Science

                                Yoshizumi Ishino, Mart Krupovic, Patrick Forterre

                                The new frontier of genome engineering with CRISPR-Cas9 by Jennifer A Doudna and Emmanuelle Charpentier.

                                ​https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/transfection-basics/gene-delivery-technologies/cationic-lipid-mediated-delivery/how-cationic-lipid-mediated-transfection-works.html​

                                ​https://www.yourgenome.org/facts/what-is-crispr-cas9​

                                Adriano Flora, PhD&Jochen Welcker, PhD

                                ​https://www.addgene.org/protocols/gel-electrophoresis/​

                                https://www.biomol.com/dateien/Bethyl--Cell-Lysate-Preparation.pdf

                                https://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf

                                http://rpdata.caltech.edu/courses/aph162/2006/Protocols/QIAquickSpin.pdf

                                https://www.takarabio.com/assets/documents/User%20Manual/Guide-it%20Mutation%20Detection%20Kit%20Protocol-At-A-Glance_111616.pdf

                                https://medicine.yale.edu/keck/ycga/microarrays/protocols/TRIZOLRNAIsolation_092107_21453_284_10813_v1.pdf

                                https://international.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i

                                ​https://www.neb-online.de/en/genome-editing/engen-mutation-detection-kit/​

                                https://bio-protocol.org/e323

                                ACKNOWLEDGEMENTS

                                I offer my deepest gratidue to Indian Academy of Science for supplementing with the wonderful opportunity. I offer sincerest thanks to my guide Dr. Manzoor Koyakutty for helping and supporting us in every step.

                                I am greatful to Ms Najma, Ms Jagathpriya and Ms Manju who always helped me in carrying out all the experiments and making me understand the protocols and science behind experiments. Lab mates Saira and Anjali also helped me a lot. My family also holds a significant place in helping me acheive this platform.

                                References

                                • https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/transfection-basics/gene-delivery-technologies/cationic-lipid-mediated-delivery/how-cationic-lipid-mediated-transfection-works.html

                                • https://www.yourgenome.org/facts/what-is-crispr-cas9

                                • https://www.taconic.com/taconic-insights/model-generation-solutions/crispr-genome-engineering-advantages-limitations.html

                                • https://www.addgene.org/protocols/gel-electrophoresis/

                                • https://www.biomol.com/dateien/Bethyl--Cell-Lysate-Preparation.pdf

                                • https://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf

                                • http://rpdata.caltech.edu/courses/aph162/2006/Protocols/QIAquickSpin.pdf

                                • https://www.takarabio.com/assets/documents/User%20Manual/Guide-it%20Mutation%20Detection%20Kit%20Protocol-At-A-Glance_111616.pdf

                                • https://medicine.yale.edu/keck/ycga/microarrays/protocols/TRIZOLRNAIsolation_092107_21453_284_10813_v1.pdf

                                • https://international.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i

                                • https://www.neb-online.de/en/genome-editing/engen-mutation-detection-kit/

                                • https://bio-protocol.org/e323

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