Molecular Genetics: Techniques Lecture Notes PDF

Molecular Genetics: Techniques 1  Four Breakthrough Technologies in Molecular Genetics 1. Recombinant DNA technology (genetic engineering): Allows to identify, isolate and combine DNA fragments (through cutting and re-attaching) from any organism to obtain the desi...

Molecular Genetics: Techniques 1  Four Breakthrough Technologies in Molecular Genetics 1. Recombinant DNA technology (genetic engineering): Allows to identify, isolate and combine DNA fragments (through cutting and re-attaching) from any organism to obtain the desired properties in a new DNA molecule that can be maintained in bacteria. 2. Polymerase Chain Reaction (PCR). Allows the amplification of small to medium-sized DNA fragments from minute amounts. Revolutionized diagnostics, forensics, isolation and cloning of DNA fragments. RNA can be amplified after copying it into DNA. 3. DNA sequencing. Allows the determination of the nucleotide sequence of a DNA fragment (and RNA after copying it into DNA). We distinguish Sanger (or dideoxy) sequencing (lower scale) from next generation and third generation sequencing techniques (large scale, high throughput as in whole genomes). 4. CRISPR/Cas genome editing. Allows the precise manipulation of DNA, and in the case of Cas13, RNA, in a wide range of organisms. The Cas9 enzyme cuts DNA in a precise location because it is guided to the target sequence through the presence of a ”single guide RNA” (“sgRNA” or simply “gRNA” for “guide RNA”). Can be used to generate simple mutations or complex gene editing (gene replacement) approaches. 2 CRISPR in the news: He Jiankui conducts unapproved genome editing in humans Parents: HIV-positive father, uninfected mother Claims to have edited CCR5 gene in embryos Introduced CCR5 allele renders cell resistant to HIV infection (if homozygous) One twin had both alleles changed, the other twin is heterozygous Unclear what happened to 14 other edited embryos Claims not validated by scientific community, since manuscript remains unpublished. Twins are probably mosaic (i.e., not all cells are resistant to HIV) International outcry, mostly because approach was uncalled for, unethical, and safety of procedure is far from certain Three years imprisonment, and fined >C$ 500,000. 3 4  Altering the genome of an organism 1. Mutation: Change the DNA sequence of a given gene to create a mutant organism. This includes both classic (random) mutations and modern (targeted) mutations 2. Transgenesis: Introduce a new piece of DNA to the genome to create transgenic organism. Typically, this adds a new protein-encoding sequence, causing the organism to produce a new type of protein that has some new property (so, in an ideal case, no genes are mutated in a transgenic organism, provided that the added sequence did not disrupt any important DNA sequences at the insertion point) 3. Gene Editing: This fairly new technique (e.g. the famous CRISPR/CAS9) is a blend of 1 & 2, depending on what exactly is done. For example, you could simply mutate a gene in a very predictable manner (1). Or, you could replace the endogenous (= normal version of the) gene with a modified version that adds a new property (1 & 2). In this case, it is a mutation (because the original gene is gone) that adds new properties that normally only a transgene could provide. 5 Simplified work flow to making transgenic or CRISPR/CAS9 organisms 1. Identify your gene of interest (GOI) 2. Isolate the corresponding GOI DNA (PCR*, or retrieve from DNA library) *PCR = Polymerase Chain Reaction 3. Clone GOI DNA (or a modified version of it) into suitable vector 4. Inject vector into host organism, so that germ cells incorporate vector DNA 5. Screen offspring for vector DNA insertion into host genome 6. Verify sequence of insertion (Validation) 7. Express transgene (e.g. Cas9 and guide RNAs to mutate GOI) 6 Simplified work flow to making transgenic or CRISPR/CAS9 organisms 1. Identify your gene of interest (GOI) 2. Isolate the corresponding GOI DNA (PCR*, or retrieve from DNA library) *PCR = Polymerase Chain Reaction 3. Clone GOI DNA (or a modified version of it) into suitable vector 4. Inject vector into host organism, so that germ cells incorporate vector DNA 5. Screen offspring for vector DNA insertion into host genome 6. Verify sequence of insertion (Validation) 7. Express transgene (e.g. Cas9 and guide RNAs to mutate GOI) 7 1. Identify your gene of interest (GOI) Typically, you are interested in a biological or disease process In mutant screens or naturally occurring mutants in a population, your attention may be directed towards a specific gene. You want to study the gene in more detail with respect to the process in question Perhaps you want to know where in the cell the protein localizes to, of if it is excreted from the cell. Or you may want to study certain parts of the protein in more detail. 8 Simplified work flow to making transgenic or CRISPR/CAS9 organisms 1. Identify your gene of interest (GOI) 2. Isolate the corresponding GOI DNA (PCR*, or retrieve from DNA library) *PCR = Polymerase Chain Reaction 3. Clone GOI DNA (or a modified version of it) into suitable vector 4. Inject vector into host organism, so that germ cells incorporate vector DNA 5. Screen offspring for vector DNA insertion into host genome 6. Verify sequence of insertion (Validation) 7. Express transgene (e.g. Cas9 and guide RNAs to mutate GOI) 9  2. Isolate the corresponding GOI DNA (PCR, or retrieve from DNA library) PCR DNA library mRNA genomic DNA mRNA genomic DNA cDNA cDNA cut DNA into manageable sizes (e.g. 10-15 kb) generate sequence-specific primers make DNA ends uniform amplify DNA in reaction tube (e.g. “polish” ends to blunt ends) clone single fragment into vector clone all fragments each into vector (one fragment per vector) 10  2. Isolate the corresponding GOI DNA (retrieve from DNA library) PCR DNA library mRNA genomic DNA mRNA genomic DNA cDNA cDNA cut DNA into manageable sizes (e.g. 10-15 kb) generate sequence-specific primers make DNA ends uniform amplify DNA in reaction tube (e.g. “polish” ends to blunt ends) clone single fragment into vector clone all fragments each into vector (one fragment per vector) 11  DNA Libraries can be used for genomic DNA or cDNAs The idea is to have an entire genome or transcriptome represented in individual plasmid DNAs (“vector DNA”) Genomic DNA is digested by restriction enzymes (usually incomplete = partial digests to generate different fragments) Restriction Enzymes recognize specific DNA sequences In the past, researchers generated their own DNA libraries. Today, one typically orders a clone corresponding to the GOI from a company or a genome project resource center image: Pierce, 6th ed. 12  Constructing a cDNA Library Cell, tissue, organism oligo-dT primer 5’ AAAAAAA-3’ 3’-TTTTTTT-5’ Reverse transcriptase 5’ AAAAAAA-3’ 3’ TTTTTTT-5’ Remove RNA 3’ Library of clones TTTTTTT-5’ representing all the DNA polymerase mRNAs in the sample 3’ TTTTTTT-5’ S1 nuclease 5’ 3’ 3’ 5’ mRNA Clone DNA into vector Transform E.coli + Plasmid vector 13 image: John Locke  2. Isolate the corresponding GOI DNA (using the Polymerase Chain Reaction = PCR) PCR DNA library mRNA genomic DNA mRNA genomic DNA cDNA cDNA cut DNA into manageable sizes (e.g. 10-15 kb) generate sequence-specific primers make DNA ends uniform amplify DNA in reaction tube (e.g. “polish” ends to blunt ends) “PCR reaction” clone single fragment into vector clone all fragments each into vector (one fragment per vector) 14  The Polymerase Chain Reaction (PCR) Nobel Prize for Chemistry in 1993 https://www.nobelprize.org/nobel_prizes/chemistry/laureat 15 es/1993/mullis-facts.html  The Polymerase Chain Reaction (PCR) What do we need? template DNA (e.g. cDNA or genomic DNA) DNA primers (short single-stranded oligonucleotides typically 17-35 bases) A heat-stable DNA polymerase all 4 nucleotides (ATP, CTP, GTP and TTP) reaction buffer reaction tube A thermocycler (contains a heatable/coolable block that holds reaction tube) Analysis of reaction: typically in a gel to visualize fragment 16 17 PCR: primers Primer sequences are given in 5’->3’ direction All DNA polymerases require primers, and attach nucleotides to the 3’ end For primers to bind to template DNA, DNA strands must be separated via heating (”melting of DNA”) Sequences that are complementary to each other will be allowed to bind to each other when cooling the temperature (typically ~50˚C to 60˚C) (the “annealing temperature”) Primers are shorter than template DNA and anneal faster than the two template strands 18 The PCR cycle 1. Melt DNA (heat to ~94 ˚C) 2. Let primers anneal (cool to ~50˚C-60˚C) 3. Let DNA Polymerase synthesize DNA (72˚C) 4. Repeat DNA doubled (after each round) 19 PCR: Repeated cycles underlie the “chain reaction” 20  Visualize your PCR results: Gel electrophoresis gels have pores through which DNA or RNA can travel The average pore size can be manipulated by changing the concentration of the gelling agent (agarose or acrylamide) DNA is negatively charged and migrates in an electric field Size separation occurs because larger fragments migrate more slowly modified from Hartwell, chapter 14, 2nd ed. 21 Simplified work flow to making transgenic or CRISPR/CAS9 organisms 1. Identify your gene of interest (GOI) (e.g. DNA encoding Cas9 or guide RNAs) 2. Isolate the corresponding GOI DNA (PCR*, or retrieve from DNA library) *PCR = Polymerase Chain Reaction 3. Clone GOI DNA (or a modified version of it) into suitable vector 4. Inject vector into host organism, so that germ cells incorporate vector DNA 5. Screen offspring for vector DNA insertion into host genome 6. Verify sequence of insertion (Validation) 7. Express transgene (e.g. Cas9 and guide RNAs to mutate gene) 22  3. Clone GOI DNA into suitable vector vector with a single EcoRI cut site Restriction Enzymes recognize specific DNA sequences PCR fragment cut with EcoRI e.g. EcoRI was isolated from E. coli and cuts 5’-GAATTC-3’ dsDNA EcoRI cuts result in 5’-overhangs called “sticky ends” Vector DNA and PCR fragment are ligate cut to make DNA ends compatible A ligase is added to fuse the PCR fragment to the vector fragment Upon successful transformation of the recombinant DNA into bacteria the DNA is said to be “cloned” 23 transform vector DNA (“plasmid”) into bacteria  Restriction Enzymes sticky end blunt end image: Pierce, 6th ed. 24 Simplified work flow to making transgenic or CRISPR/CAS9 organisms 1. Identify your gene of interest (GOI) (e.g. DNA encoding Cas9 or guide RNAs) 2. Isolate the corresponding GOI DNA (PCR*, or retrieve from DNA library) *PCR = Polymerase Chain Reaction 3. Clone GOI DNA (or a modified version of it) into suitable vector 4. Inject vector into host organism, so that germ cells incorporate vector DNA 5. Screen offspring for vector DNA insertion into host genome 6. Verify sequence of insertion (Validation) 7. Express transgene (e.g. Cas9 and guide RNAs to mutate gene) 25 P-Element transformation in Based on a transposable element Drosophila (example for a transgene) (“Transposon”, here: P-Element) The Enzyme integrating the transposon DNA into new integration sites is the “Transposase” Only the helper plasmid encodes a functional copy of the Transposase gene. The actual P-Element (the vector with recombinant DNA) contains recognition sites for the Transposase Both, the P-element vector and the helper plasmid are co-injected into the fly embryo, leading to insertion of the P- element (but not the helper) into the host genome. Successful integration can be monitored by expression of the w+ gene (makes red eyes), because the host genome is w- (has white eyes) 26 Simplified work flow to making transgenic or CRISPR/CAS9 organisms 1. Identify your gene of interest (GOI) (e.g. DNA encoding Cas9 or guide RNAs) 2. Isolate the corresponding GOI DNA (PCR*, or retrieve from DNA library) *PCR = Polymerase Chain Reaction 3. Clone GOI DNA (or a modified version of it) into suitable vector 4. Inject vector into host organism, so that germ cells incorporate vector DNA 5. Screen offspring for vector DNA insertion into host genome 6. Validation: Verify sequence of insertion (or mutation if using CRISPR in 7.) 7. Express transgene (e.g. Cas9 and guide RNAs to mutate gene) 27  Sanger Sequencing Frederick Sanger won Nobel Prize for Chemistry in 1958 and 1980 Prize in 1980 was shared with Walter Gilbert, who had developed a different DNA sequencing technique His approach to DNA sequencing is still widely used today (in optimized and automated form). We will discuss the original and the modern versions. 28  Sanger Sequencing Use a DNA Polymerase to copy any given (Dideoxy Sequencing) fragment of DNA (say a PCR fragment representing your modified gene you want to insert using CRISPR/Cas9) Use normal nucleotides (dNTPs) to allow DNA synthesis by the DNA polymerase The reaction also contains a small percentage of special nucleotides (ddNTPs) that, once incorporated, will block further strand extension from that position onward. ddNTPs lack the 3—OH group required by the Polymerase to add new nucleotides, but are otherwise handled like normal dNTPs by the Polymerase Think of ddNTPs as randomly incorporated roadblocks, and the result is a population of fragments where all strands end with a “T” in one reaction, and with an “A” in another reaction, and so on. The fragments can be separated with single Pierce, 6th ed. nucleotide resolution 29  Two types of Sanger sequencing exist: Original and Automated The original approach (see figure) used four different reactions, one each for ddATP, ddCTP, ddGTP and ddTTP all fragments in the ddATP- containing reaction tube ended with “A”, the other tubes correspondingly with C, G, and T The fragments were all radioactively labelled, because the reaction was “spiked” with a small percentage of radioactive dNTP. The resulting fragments are denatured, and single strands are separated on a polyacrylamide gel that allows single-base pair resolution. 30 Pierce, 6th ed.  Automated Sanger Sequencing uses ddNTPs that are each labelled with a different fluorescent dye. Normal dNTPs are present as well The reaction takes place in a single reaction tube After the reaction, the DNA is denatured and single strands are separated, followed by detection of the fluorescent dyes as the fragments migrate through. 31 modified from Hartwell, 2nd ed.  Automated Sanger Sequencing 32 modified from Hartwell, 2nd ed. Simplified work flow to making transgenic or CRISPR/CAS9 organisms 1. Identify your gene of interest (GOI) (e.g. DNA encoding Cas9 or guide RNAs) 2. Isolate the corresponding GOI DNA (PCR*, or retrieve from DNA library) *PCR = Polymerase Chain Reaction 3. Clone GOI DNA (or a modified version of it) into suitable vector 4. Inject vector into host organism, so that germ cells incorporate vector DNA 5. Screen offspring for vector DNA insertion into host genome 6. Verify sequence of insertion (Validation) 7. Express transgene (e.g. Cas9 and guide RNAs to mutate gene) 33  What is CRISPR? Stands for Clustered Regularly Interspaced Short Palindromic Repeats Discovered in bacteria and archaebacteria Serves as a memory against phages DNA from invading phage DNA is stored in a specific locus (CRISPR) of the bacterial chromosome Should another infection occur from a phage with the same DNA, the stored CRISPR DNA is used to identify the invading DNA, and cut it via Cas (CRISPR-associated proteins) nucleases. In other words, Cas nucleases are DNA-degrading enzymes and guided to their target by CRISPR-derived RNA. 34  2. Gene Editing: CRISPR/Cas9 Idea: Recruit a DNA-cutting enzyme (“Nuclease”) to an exactly specified locus in the genome (with single nucleotide precision). These are called “engineered nucleases”. The nuclease cuts the DNA, creating a double-strand break In the simplest version of CRISPR, let the cell repair the break via NHEJ (Non- homologous end joining) NHEJ will cause small deletions in the targeted region => the gene will be mutated The more advanced version of CRISPR exploits homologous recombination (HR), also known as Homology-Directed Repair (HDR). Normally, HR can occur when replication has occurred and an identical copy is present in the cell to repair the break. In advanced CRISPR, one injects donor DNA that is homologous (but not identical) to the region with the double-strand break. This ”fools” the HR system to use the injected donor DNA rather than the endogenous copy it would normally use. CRISPR/Cas9 can be used both in the germline (all cells in the affected F1 offspring will be modified) or selectively activated in specific cells of an organism (creating chimaeras). The latter requires organisms that carry Cas9 and gRNA transgenes in all cells, where one of the two components is activated in a cell-specific manner. 35 36 Cas9 is an RNA-binding Nuclease The single RNA that is incorporated into the CAS9 enzyme serves as a guide to direct the CAS9/RNA complex to DNA that is complementary to the RNA. The RNA is therefore called ”guide RNA” (gRNA) or “single guide RNA”, (sgRNA) Only a specific part of the RNA is used to find the corresponding DNA The gRNA is designed by the scientist, so that a specific gene is targeted The PAM sequence is a short sequence (e.g. NGG in many bacteria) that needs to be present next to the target sequence for initial binding of the Cas9 effector complex 37  The two DNA repair pathways exploited by CRISPR/Cas9 (= donor DNA) I. Small imprecise deletions II. Large defined deletions or customized genome editing new region of DNA not previously present in host organism Hsu et al., 2014 38  Cutting with Precision Gene of Interest (GOI) CRISPR video modified after Gratz et al., 2013 39  What makes CRISPR/Cas9 so powerful? Advantages: could be potentially used in virtually all organisms unprecedented precision in altering the genome allows for fast, simple mutations in gene of interest allows for fast gene editing strategies to replace endogenous gene with virtually any sequence high potential for gene therapy Dangers/disadvantages: Requires us to rethink ethical standards. Should we fix severe genetic errors in early human embryos? What about not so severe problems? Should we make us, as a species, smarter, less aggressive, less sad, less greedy…. less human?? A brave new world? The “CRISPR gene drive”, a special form of a self-copying CRISPR-modified locus can be used to suppress or even eradicate (in theory) entire species. Do we have the right to wipe out mosquitos from the face of the earth? Non-specific DNA cuts by Cas9 40 Restoring sight to blind mice via CRISPR/Cas9 41