The CRISPR Revolution/ The Synthetic DNA Revolution

Application Session 3 The CRISPR Revolution/ The Synthetic DNA Revolution o Up until the last 10 years the methods of changing genomic DNA in a specific way (e.g. deleting/modifying a gene, fixing a gene) has been: o Slow: waiting for rare, mostly uninduced DNA repair events to initiate the process ▪ Can induce site-specific repair, but using protein-guided endonucleases o Technically challenging: producing “repair templates” through recombinant DNA technology o But two technical advances have changed the game. o CRISPR: an RNA-guided endonuclease to cause double strand breaks (DSBs) at the genomic site you want; rapidly reprogrammed by using a different, single guide RNA (sgRNA) o Synthetic DNA: you can inexpensively design and order whatever sequence of DNA you want Clustered regularly interspaced short palindromic repeats o Bacteria and archaea evolved this system in order to recognize and destroy invading phage DNA o After an infection, part of the phage DNA is incorporated into the host’s DNA in a CRISPR array o When the Cas (CRISPR associated) genes are transcribed, so are the small phage DNA sequences o These sequences can then target and destroy complimentary DNA The power of CRISPR/Cas9 lies in the ability to reprogram it with an RNA molecule Nature: use 2 RNAs that hybridize Biotech: fuse 2 RNAs into 1 single guide RNA (sgRNA) These sgRNAs can be customized by researchers to target genes-of-interest The RNA of the RNA/Protein complex guides where it should bind to the genomic DNA (or invading viral DNA) o The protein (Cas9) introduces a double strand break there o DSB is repaired using homologous recombination (HR) or non-homologous end joining (NHEJ) o o o o What can you do with CRISPR + Synthetic DNA? o What can we do… right now: o You can cut and modify whatever genomic locus you want, in whatever organism you want. Simply put, you can now: o Order the single guide RNAs (sgRNAs)that dictates the target o Order the repair template (dsDNA) that introduces the change you want o Cure genetic diseases in model organisms (cataracts in mice) o Edit disease associated genes: Cystic Fibrosis (CF), Beta-thalassemic o Make a “low gluten” version of wheat o Edit human T-cells ex vivo, reintroduce into the patient to target blood cancer o Genetically modify human embryos UNIT 20 Cell fractionation by centrifugation o You can separate different cellular components from each other based on their size (density) o The smaller the particle, the faster you need to spin to “pellet” it o Many proteins have defined locations in cells (mitochondria, ER, Golgi, nucleus, etc.) o A centrifuge spins tubes and the force on the contents of the tubes varies with the speed of rotation o Organelles and macromolecules can be separated by centrifugation How can gradients allow for separation in a centrifuge? o Small and large biomolecules can be separated based on their mass and shape by special types of gradients o Sucrose o Cell types can be separated from other cell types too o Percoll Properties of Amino Acids enable protein purification o Composition of amino acids affects the ability of proteins to stick to different surfaces (resins) and this is exploited to separate proteins from each other How does it work? – Ion Exchange (IEX) Chromatography o Proteins are composed of unique numbers and combinations of amino acids o Some have more acidic amino acids, giving them a stronger negative charge o Others are rich in basic amino acids, giving them a more positive charge o Some amino acid side chains are charged o This allows proteins to bind with beads that have the opposite charge of their accessible/exposed amino acids o Changing pH o Using salt gradient How does it work? – Gel filtration / Size Exclusion Chromatography o There is a large range in the size of proteins, from very small (few amino acids) to very large (1000’s of amino acids) o Can separate based upon size by using beads with pores o Large molecules flow through faster, and small molecules flow through slower How does it work? – Affinity Chromatography o o o o Proteins that have an affinity for a ligand Attach the ligand to the beads Capture protein that can bind to the ligand, wash away the rest Elute off the protein by breaking the interaction between the protein and its ligand o Protein purification involves successively passing fractions containing the protein activity over different columns in sequence o Each step purifies the sample and removes more contaminating proteins… but… you lose material at every step Why not make purifications “standardized” to use the same approach? o What kind of epitope tags do molecular biologists commonly put on the N- or C- termini? o How do you capture proteins by that tag? o His-His-His-His-His-His (6xHis) o Nickel-NTA-Agarose o Glutathione-S-Transferase (GST) o Glutathione-Agarose o Maltose binding protein (MBP) o Amylose-Agarose o Protein A o IgG Antibody-Agarose o Calmodulin Binding Peptide (CBP) o Ca++/Calmodulin-Agarose So, if one epitope tag is good, why not use more than one? o Protein-of-interest has 2 tags: o Protein A o Calmodulin-binding peptide o Protein A binds to IgG on a bead o Elute proteins from the beads by cutting them off with a protease o Calmodulin-binding peptide binds to calmodulin on a bead o Elute proteins from the beads by removing Ca2+ with EGTA UNIT 21 Gel Electrophoresis o Various types of gel electrophoresis are used to characterize preparations of biomolecules (typically proteins or nucleic acids) o Negatively charged molecules will move from top to bottom (minus to plus) o Mobility will be affected by size and shape because the biomolecule collides with the gel matrix SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) o A method to characterize the composition of samples that separates molecules based upon their mass, not their shape or charge o Two key reagents for SDS-PAGE: ▪ Sodium dodecyl sulfate (SDS) does two things: • SDS denatures (unfolds) proteins • SDS coats the polypeptide chain and gives it a negative charge ▪ Beta-Mercaptoethanol breaks disulfide bonds How can we see proteins on a gel? o Dyes such as Coomassie blue and solver stain can allow us to see proteins But what if you don’t know what protein is important in a complex mixture? How do you determine what all of the proteins are? Can you “sequence” proteins? o Cut proteins into smaller units by using a site-specific protease trypsin: cuts before R or K o Peptides can be separated by column chromatography o Peptides are then fragmented and the resulting pieces can be used to identify the order of amino acids present, and post-translational modifications o Antibodies are used in many assays for proteins because antibodies can be selected that bind specific proteins o Antigen: the molecule used in the immunization (e.g. protein, nucleic acid, small molecules) o Epitope: the part of the antigen that is recognized by an antibody Raising specific antibodies to a protein-of-interest o Antibodies are proteins produced by the vertebrate immune system that recognize “foreign” molecules which have entered the body o Monoclonal antibody: an isolated antibody that binds a single epitope on one antigen o Polyclonal antibodies: a mixture of antibodies that together bind many epitopes to one antigen UNIT 22 Manipulating DNA: Molecular Cloning o Often called “recombinant DNA technology”, “genetic engineering”, or “DNA cloning” o “Cut-and-Paste with DNA”: o DNA molecules can be cut into fragments with restriction endonucleases ▪ Bacteria produce these nucleases to protect themselves against invasive DNA ▪ Restriction enzymes bind a specific DNA sequence, cut both strands, and can leave blunt ends or ends with an overhang ▪ Hundreds of restriction nucleases are available o DNA fragments with compatible ends can be ligated together in the test tube (the “paste” portion of cut-and-paste) o The products of the ligation are called recombinant DNA molecules Generation of a recombinant DNA molecules o In this example, a foreign piece of DNA is ligated into a circular DNA called a plasmid o The plasmid contains an origin of replication that allows it to be replicated in bacteria What gene do you want to clone? How do you get that DNA? o Libraries: a collection of all nucleic acid sequences in a genome o Genomic DNA (gDNA) libraries ▪ All DNA in genome; regulatory regions, introns etc. o Complementary DNA (cDNA) libraries ▪ Complementary to RNA, expressed sequences in the genome o o o o Gel electrophoresis is used to characterize DNA Large DNA migrates more slowly than small DNA DNA is usually detected in the gel with dye or radioactive label Gel electrophoresis is often used to isolate specific DNA fragments and to characterize a population of DNA molecules produced in an experiment o DNA fragments are often separated on agarose gels o Agarose is a polymer of carbohydrate o Like separating proteins in PAGE gels, the smaller fragments move faster through the matrix Nucleic acid hybridization can be used to detect rare specific sequences among many in a complex mixture o Southern blotting = detect DNA o Northern blotting = detect RNA o Nucleic acid hybridization: o Highly specific binding between two strands with sequence homology. This is how nucleic acids are detected in complex mixtures: using a labeled probe Polymerase Chain Reaction (PCR) revolutionized genetic engineering o PCR allows us to… o Amplify and isolate minute amounts of DNA o Clone genes o Sequence DNA o Modify DNA sequences (adding nucleotides, making mutations, etc.) Polymerase Chain Reaction (PCR) is a common way for isolating a rare specific DNA fragment from a mixture o The scientist selects the primers, so some sequence information is needed. This information is readily available for organisms with sequences genomes o PCR can be used to amplify sequences of a gene o Template for the PCR amplification can be… o DNA: genomic clone o RNA: complementary DNA (cDNA) clone UNIT 23 A DNA sequence can be determined using dideoxy nucleotide “chain terminators” o Dideoxy sequencing uses DNA polymerase, along with special chain-terminating nucleotides called dideoxy ribonucleoside triphosphate, to make partial copies of the DNA fragment to be sequenced o Fully automated machines can run dideoxy sequencing reactions o The automated method uses an excess amount of normal dNTPs plus a mixture of four different chain-terminating ddNTPs, each of which is labeled with a fluorescent tag of a different color o Shotgun sequencing: to determine the nucleotide sequence of a whole genome, the genomic DNA is first fragmented into small pieces and a genomic library is constructed, typically using plasmids and bacteria Next (2nd ) generation sequencing: illumina platform o RNA-sequencing (RNA-seq): o Isolate mRNA o Convert to cDNA (by RT enzyme) o Sequence as described above o Strand-specific sequencing What direction is your screen? o Forward screen: broadly change the genes, and select the phenotype you want the study o Chemical mutagen: hit the genome with a DNA-damaging agent o Transposable elements: pieces of DNA that can insert semi-randomly with the help of proteins encoded by it o Natural sequence variations: sequence many members of a population (e.g.) and correlate with disease traits o Reverse Screen: select the gene you want to modify, and broadly observe for phenotypes that may result o Introduce a mutation: integrate a different copy of the gene in the normal genomic locus o Delete/ Knock Out (KO) the gene: integrate a piece of DNA lacking the gene into the normal genomic locus o Add another copy of the gene: integrate the correct or variant gene into a different genomic locus o Knock down (KD) the mRNA or protein: use methods to cause specific mRNA or protein degradation UNIT 24 Isolating cells and growing them in culture o Populations of cells can be grown in culture dishes o Can treat cultured cells in ways that are not possible with whole animals o Most vertebrate cells isolated from tissues have limited proliferative potential (they can be kept for a short period of time)- these are called primary cells or cell strains o Many cell lines are derived from cancer cells because these proliferate indefinitely – tis results from genetic changes The type of microscopy required is dictated by the size of the structure o Diameter of a typical animal cell – 10 to 20 microns o Micro = micrometer = um = 0.001 mm o The head of a pin is about 1 mm in diameter so a cell is about 1/100 the diameter of a pin head o Diameter of the ribosome – 20 nanometers o Diameter of atom or length of a chemical bond – C-C: 0.154 nanometers = 1.54 angstroms Conventional Light Microscopy o To see something, the specimen must disturb the incident light in some way, such as… o The amount of light o The path of the beam Contrast: light interference o To observe a specimen, it must… o Absorb particular wavelengths of light (which results in color) o Slow the light so that different paths of light are out of phase Bright-Field Microscopy o Bright-field Microscopy is poor for viewing colorless samples such as most animal cells Phase-contrast microscopy o The optics of a phase-contrast microscope uses the shift in the phase of light as it passes through neighboring parts of the cell to enhance contrast Visualizing cells from a tissue/organ o This sample of ducts in the kidney has been: o Fixed to preserve its structure o Thinly sliced so that it transmits light, and o Stained so that it can be seen by bright field microscopy o “stains” are chemical dyes that bind tightly (or more intensely) to certain biomolecules o Stains provide color or contrast compared to the surrounding areas of the cell/tissue o Light microscopy lacks the resolution for a detailed view of cellular compartments o Transmission electron microscopy (TEM) provides the needed resolution Transmission Electron Microscopy o o o o o Practical resolution for biological samples can be 2A now… atomic resolution Radiation beam is electrons, which have very short wavelengths. Higher resolution The lenses are magnets that focus the electron beam An image is created when atoms can scatter electrons The small atoms of life (C,H,O,N,P,S) do not scatter electrons very well o So, samples are almost always stained with heavy metals such as uranium, lead, platinum, osmium or gold since these do scatter electrons o The samples are dead and fixed Scanning Electron Microscopy (SEM) o Samples still are fixed o Resolution is not as high as TEM o But SEM can provide 3D images of structures, while TEM can only do 2D UNIT 25 Fluorescence Microscopy o Blue = DAPI fluorescence from the dye bound to DNA o Red = a fluorescent antibody bound to a centromere protein o Green = a fluorescent antibody bound to tubulin Why use fluorescence microscopy? o Highly selective fluorescent tags allow us to determine the location of specific molecules in a cell o Fluorescently tagged molecules can be introduced into cells and their behaviors can be viewed in a live cell o Combining different fluorescent tags can allow you to show two proteins are located in the same place and might work together Indirect Immunofluorescence Assay (IFA) o The molecule-of-interest is “fixed in place in the cell with a chemical crosslinker (formaldehyde) o The cell is then permeabilized with detergent to allow molecules to get into the cell o The molecule-of-interest is bound by a “primary antibody” o The primary antibody is bound by a fluorescently labeled “secondary antibody” to permit detection