Additional Material.docx

5.1. Vaccine Production Traditional method: making recombinant gene to produce recombinant protein as vaccine Kariko & Weissman: discovered that base-modified mRNA can be used to block activation of inflammatory reactions mRNA vaccines: delivery via lipid nanoparticles Allows transport through...

5.1. Vaccine Production Traditional method: making recombinant gene to produce recombinant protein as vaccine Kariko & Weissman: discovered that base-modified mRNA can be used to block activation of inflammatory reactions mRNA vaccines: delivery via lipid nanoparticles Allows transport through cell membranes Protects RNA from being broken down Moderna and Pfizer: used lipid nanoparticles with PEG (which make them last longer in the body) Limitation: need for low temperature Bivalent vaccines Advantage: you can make different variants of mRNA very rapidly mRNA vaccines for other diseases currently in clinical trials 5.2. Genome editing Eukaryotic model organisms Saccharomyces cerevisiae Drosophila melanogaster Caenorhabditis elegans Mus musculus Knockout mice Studying of genetic diseases and efficacy of treatments Classic ways of genome editing Site-directed mutagenesis Cassette mutagenesis Use of viral vectors Site-directed mutagenesis PCR-based technique of changing DNA at a desired position e.g. SNP, missense, deletion, etc. Determines effect of DNA sequence change on function Example: site-directed modified subtilisin have reduced activities Cassette mutagenesis Variety of mutations already introduced to gene of interest Use of viral vectors Random (anywhere in the genome) vs targeted insertion 5.3. Analysis of Gene Function RNAi or post-translational gene silencing Produces siRNAs that silence complementary sequence Degradation of mRNA blocks gene functions Function: immune response against foreign RNA Synthetic siRNAs Limitation: only produces atransient outcome as it only targets mRNA, allowing continuous transcription of the DNA sequence; siRNA-based treatments should be continuous Gene disruption/knockout Mutated gene is introduced to embryonic stem cell Normal gene “knocked out”by foreign gene via homologous recombination Gene knock-in Genome editing vs genetic engineering Genome editing: in situ alteration of DNA sequence DSB DNA repair mechanisms: NHEJ and HDR Genome editing mechanisms Zinc finger nucleases (ZFNs): zinc finger DBD anchored to engineered nuclease FokI which makes a nonspecific DS break TALENS gene editing - single nucleotide resolution: TALENs DBD (have longer recognition sequences than ZFNs) anchored to FokI CRISPR Variants: can induce gene silencing or enhance transcription Medical applications (gene knockout) Industrial applications Genome-edited crops Gene drives Systems of biased inheritance Potential uses: population suppression or replacement Gene drive mosquitoes engineered to fight malaria will pass resistance to Plasmodium to its offsprings Overview RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated. This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used to suppress genes that are overactive in diseases such as cancer. The Process First, researchers synthesize double-stranded RNA with a sequence complementary to the targeted gene. Different types of double-stranded RNA can be used, including small interfering RNA (siRNA) and short hairpin RNA (shRNA). shRNA is one strand of RNA that is folded over—creating a double-stranded RNA with a hairpin loop on one side—and is a precursor of siRNA. The double-stranded RNA is then introduced into cells by methods such as injection or delivery by vectors, such as modified viruses. If shRNA is used, RNase enzymes in the cell, such as Dicer, cleave it down to the shorter siRNA, removing the hairpin loop. The siRNA then binds to an enzyme Argonaute, which is part of a complex called the RNA-induced silencing complex (RISC). Here, the two strands of the siRNA separate. One floats away while the other—called the guide strand—remains attached to the RISC. It is known as the "guide strand" because it is the strand that binds the mRNA through complementary base pairing, bringing the RISC to the mRNA. This binding is very specific because the siRNA is usually designed to be completely complementary to the targeted mRNA. Argonaute then cleaves and degrades the mRNA, preventing it from being translated into protein— effectively silencing the gene. Procedure In RNA interference, or RNAi, small non-coding RNAs bind to complementary messenger RNAs, mRNAs, to prevent their translation to proteins. Three classes of small RNAs can accomplish RNAi— microRNAs or miRNAs are encoded by a cell's genome; small interfering or siRNAs are derived from exogenous viral double-stranded RNAs; and PIWI-interacting or piRNAs, are specific to the germline. In general, the RNAi pathway begins when the enzyme Dicer cleaves double-stranded RNA to an approximately 20-25 nucleotide-long siRNA. siRNA then binds to a group of proteins called the RNA-induced silencing complex, RISC. In RISC, the RNA guide strand remains in the complex while its complementary strand is removed. The guide strand then binds to its complementary sequence in mRNA. Argonaute, an enzyme that is part of RISC, then cleaves the mRNA, thereby silencing the targeted gene. Overview A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of “genome editing”, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage, mutations can be introduced into the targeted DNA region. In this video, JoVE explains the principles behind genome editing, emphasizing how this technique relates to DNA repair mechanisms. Then, three major genome editing methods—zinc finger nucleases, TALENs, and the CRISPR-Cas9 system—are reviewed, followed by a protocol for using CRISPR to create targeted genetic changes in mammalian cells. Finally, we discuss some current research that applies genome editing to alter the genetic material in model organisms or cultured cells. Procedure Genome editing comprises techniques with which researchers can “edit” or change a specific DNA sequence. These methods rely on the creation of small “cuts” in DNA, which cells attempt to repair, often incompletely. In this manner, scientists can induce mutations in targeted sequences with greater efficiency compared to classical gene targeting. In this video, we will review the principles behind three genome editing techniques, and discuss a generalized protocol for one of them, the CRISPR-Cas9 system. We will then explore some applications of these methods. First, let’s look at the principles behind genome editing. The classical method for introducing genetic changes to specific sequences in the genome is gene targeting by homologous recombination, where the incorporation of homologous sequences into the targeting construct leads to the “switching out” of the endogenous gene for an altered version. Like gene targeting, genome editing can target changes to specific DNA regions, but it can do so with much higher efficiency, establishing the desired mutations in more cells in any given experiment. All genome editing methods rely on a cell’s ability to repair double-strand breaks made to its DNA by endonucleases. This damage may be repaired by nonhomologous end joining or NHEJ, where broken DNA ends are resealed directly; or by homology-directed repair or HDR, where the damage is fixed by copying from a homologous template. NHEJ is not usually perfect, and could result in several bases being deleted or added to the repaired DNA, thus mutating the target sequence. On the other hand, HDR allows researchers to add in a template to direct specific changes to the target site. Three major genome editing techniques have been developed and popularized over the last few years. In one method, researchers fuse DNA-binding “zinc finger” domains of certain transcription factors to the DNA-cleaving domain of the FokI endonuclease. As each zinc finger domain recognizes a specific nucleotide triplet, these domains can be artificially linked together to engineer zinc finger nucleases, or ZFNs, that target unique DNA sequences. Similarly, nucleases called “TALENs” join FokI to the variable DNA-binding domains of bacterial proteins called transcription activator-like effectors, or TALEs. Each TALE domain recognizes a single, unique DNA base, giving TALENs their sequence specificity. Finally, the CRISPR-Cas9 system uses components of a prokaryotic immune system that normally protects its host against incursion by foreign genetic materials, like those in viruses or plasmids. In this system, pieces of invading foreign DNA called “protospacers” are incorporated into a CRISPR locus, which is then transcribed and processed by protein-RNA machinery into small RNAs called crRNAs. Scientists are harnessing the CRISPR machinery for genomic editing purposes by designing crRNA-like guide sequences known as “single guide” or sgRNA, which can be used to target Cas9 to almost any desired genetic sequence in mammalian cells, or other systems of interest. The simple customizability of the RNA sequence-based CRISPR-Cas9 system provides distinct advantages over protein-based genome editing methods like ZFNs and TALENs. Now that you’ve learned about the principles behind genome editing, let’s review a typical protocol of using CRISPR to create targeted genetic changes in mammalian cells. First, a genomic location to be edited is chosen. This region must be short—approximately 20 base pairs in size—and possess a 3-basepair long “protospacer adjacent motif” or “PAM” at its 3′ end. The PAM is required for Cas9 to recognize and cleave the target DNA region, and the exact sequence is specific to the organism-of-origin of the Cas9 being used. Once a potential site has been identified, it should be searched against the genome sequence to ensure that similar sites are not found elsewhere in the genome, and thus no off-target genomic regions will be edited. To synthesize the CRISPR guide sequence, two oligonucleotides are generated, one identical and one complementary to the target sequence. Extra sequences are included on their 5′ ends for compatibility with the sgRNA vectors. These oligonucleotides are then mixed together, denatured, and then annealed. Next, a plasmid containing an sgRNA “scaffold” is cut with the appropriate restriction enzyme, mixed with the synthesized CRISPR guide sequence, and incubated with ligase enzyme to create the sgRNA construct. The plasmid can then be transformed into bacteria and cultured for amplification. Once purified from bacteria, the CRISPR construct is co-transfected with a construct encoding Cas9 into the cells whose genome is to be edited. These transfected cells are cultured to form clonal colonies. Genomic DNA can be isolated from each colony and analyzed by PCR or sequencing to screen for those in which the CRISPR system has produced the desired mutations. Let’s now look at some ways scientists are using genome editing techniques. Many researchers use genome editing to introduce reporter sequences into specific loci. In this experiment, ZFNs were used to insert green fluorescent protein into a gene that is involved in neuron development and survival. Researchers confirmed that they correctly targeted this gene by directing stem cells to differentiate into neurons, and performing double immunofluorescence for GFP and neuronal markers. Genome editing has also made it much easier to generate “knockout” organisms in which a gene of interest is rendered non-functional. Here, researchers sought to create knockout mice by injecting embryos with mRNA encoding TALENs that target specific genes. Subsequent endonuclease treatment of DNA from TALEN-treated mice allowed researchers to identify animals with mutations in both copies of the targeted gene. Finally, genome editing can be used to create large genetic deletions by causing two double-strand breaks in the same chromosomal region. These large deletions might be necessary when the function of a gene or genetic element cannot be knocked out by the small deletions created with conventional genome editing protocols. Here, researchers co-electroporated mouse cancer cells with a GFP construct and two CRISPR constructs targeting different areas within a cancer-driving gene. Cells that were successfully transfected, which would thus emit GFP signal, were isolated by fluorescence-activated cell sorting, and subsequent PCR and sequencing identified cells in which the DNA region between the two CRISPR-targeted sites was deleted. You’ve just watched JoVE’s video on genome editing. Here, we’ve reviewed the principles behind ZFNs, TALENs, and CRISPR, explored a general CRISPR-Cas9 protocol, and discussed how researchers are using genome editing techniques today. As always, thanks for watching! Overview Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9. A basic CRISPR-Cas9 system consists of a Cas9 endonuclease and a small RNA that guides Cas9 to the target DNA. Origin CRISPR sequences were first observed in bacteria and later identified in archaea. Researchers discovered that the CRISPR-Cas9 system serves an adaptive immune defense against invading viruses. Many bacteria and most archaea capture short sequences of the viral DNA to create a library of virus DNA segments, or CRISPR arrays. When the prokaryotes are re-exposed to the same virus or class of viruses, CRISPR arrays are used to transcribe small RNA segments that help recognize viral invaders and subsequently destroy viral DNA with Cas9 or a similar endonuclease. Using CRISPR-Cas9 Technology CRISPR-Cas9 is commonly used in the laboratory to remove DNA and insert a new DNA sequence in its place. To achieve this, researchers must first create a small fragment of RNA called the guide RNA, with a short sequence called the guide sequence that binds to a specific target sequence on genomic DNA. The guide RNA can also associate with Cas9 (or other endonucleases like Cpf1). The guide RNA and Cas9 protein are administered to a cell of interest where the guide RNA identifies the target DNA sequence and Cas9 cleaves it. The cell’s machinery then repairs the broken strands by inserting or deleting random nucleotides, rendering the target gene inactive. Alternatively, a customized DNA sequence may be introduced into the cell along with the guide RNA and Cas9, that serves as a template for the repair machinery and replaces the excised sequence. This is a highly effective way for researchers to “knock out” a gene to study its effects or replace a mutated gene with a normal copy in hopes of curing a disease. Ethical and Feasibility Considerations in Humans As a result of the significant gene modification capabilities of the CRISPR-Cas9 system, there has been great debate over its use, especially in regards to embryo editing. A Chinese scientist recently claimed to have created genome-edited babies using CRISPR technology to disable a gene involved in HIV infection. This led to a global outcry from scientists concerned about the ethical and safety considerations of the procedure. Many have called the move premature, and others have expressed concerns over off-target genomic effects. While the number of possible biotech applications for the CRISPR-Cas9 system is numerous, it is important to consider future challenges that may arise as a result of its use. Procedure The CRISPR-Cas9 system is a DNA editing tool that stands for, Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein 9. First observed in bacteria, CRISPR-Cas9 is a means of defense against viruses. As foreign viral DNA enters a bacterium, it's processed into smaller fragments, which may be inserted into a region of the bacterial genome called a CRISPR Locus. When the region is transcribed, the product associates with smaller RNAs called tracrRNAs which may help to orient both the Cas9 protein and RNAse to the molecule. The latter of which cleaves the transcript. The end result is several complexes each consisting of a Cas9 protein, tracrRNA and a crRNA derived from DNA in the Locus. The CRISPR RNA in these structures recognizes and guides Cas9 to viral DNA which is then cleaved and destroyed. Scientists harness CRISPR-Cas9 by synthesizing individual RNA molecules that mimic tracrRNA and CRISPR RNA which can target a gene of interest. For example when two such guide RNAs are introduced into cells with Cas9 and both target the same gene, a sequence can be excised. Once this target region is removed the cut ends are reconnected and the effects on the cells are observed. Thus the CRISPR-Cas9 system is modified from a bacterial mechanism. And can be employed for an array of gene editing techniques.