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Part 16
Genome Editing
| Question/Answer | Term/Definition |
|---|---|
| Genome Editing | Allows scientists to introduce targeted changes to the DNA of an isolated cell or an entire organism. For example, scientists can insert a DNA sequence, delete a DNA sequence, or modify the DNA sequence of any gene via genome editing. |
| What is the goal of genome editing? | The goal of genome editing is to change the phenotype of a cell in a way controlled by the researcher. |
| What is the application of genome editing? | The applications of genome editing are staggering. Genome editing can be used in research to better understand the role of a gene and its protein product in cellular structure and function. |
| What can genome editing also be used for? | Genome editing can also be used as a treatment for genetic diseases by replacing a mutant gene that causes the disease with the normal (wild-type) version of the gene. |
| What is the last way genome editing can be used? | Finally genome editing can be used to enhance the yield of crops or give desirable traits to livestock. |
| How does genome editing work (1) | Genome editing works by recognizing a specific target DNA sequence in the genome. After recognition of the target DNA sequence an endonuclease cuts bothe DNA strands. |
| What does the cell do when the DNA strands are cut? in genome editing | The cell then tries to fix the double-stranded DNA break by rejoining the two ends of the severed DNA molecule; however the repair mechanisms involved are error-prone, introducing extra nucleotides or deleting nucleotides at the cut site. |
| What happens with the insertion or deletion of a single base in genome editing? | Insertion or deletion of a single base or two bases within the coding region of a gene changes every codon downstream of this insertion/deletion(indel) site. This type of mutation, referred to as a frameshift mutation produces a defective protein product. |
| What are the three major genetic technologies that can be used to edit DNA sequences within isolated cells or entire organisms? | Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9. |
| How are ZFNs used? | ZFNs are enzymes engineered in the lab to contain two parts: a zinc-finger motif and a endonuclease. The zinc-finger motif allows the ZFN to bind to the target DNA sequence. One ZDN attaches to one DNA strand about ten base pairs away. |
| What happens in the ZFN process when the endonucleases cut? | The endonucleases come together and cut both DNA strands between the ZFN binding site. Because the ZFN binds and then cuts a specific target DNA sequence, ZFNs only create a single genome edit at a time |
| How are TALENs used? | Like ZFNs TALENs are enzymes designed by researchers to include both a DNA-binding region and a endonuclease region. The TALEN DNA-binding protein domain can be engineered to bind to any target DNA sequence. |
| What happens when endonuclease binds to the DNA is the TALEN process? | Once bout to the DNA the TALEN endonuclease domain cuts both strands of the target DNA sequence, allowing the creation of a single genome edit at a time. |
| How is a CRISPR-Cas9 used? | The CRISPR-Cas 9 system is the newest, most powerful and versatile genome editing technique. CRISPR-cas9 can be used to create a single genome edit or multiple genome edit simultaneously. |
| What is something about ZFNs and TALENs? | ZFNs and TALENs have many drawbacks, including the high cost and time involved in engineering the DNA binding domains within the nucleases and the inefficient cutting of the target DNA sequence. |
| Why is CRISPR-Cas9 better than ZFN and TALEN? | Although ZFN and TALEN have been used to successfully edit genes, the science world has embraced CRISPR-Cas9 due to its lower cost, higher efficiency, and potential to create multiple genome edits simultaneously. |
| What is cool about the CRISPR-Cas9? | It is currently used all over the world and has a promising future. |
| What CRISPR and acronym for? | CRISP is an acronym for the clustered regularly interspaced short palindromic repeats (CRISPR) system. It has two molecular components. |
| Describe the first CRISPR-Cas9 molecular component. | A single guide RNA (sgRNA) consists of a single stranded RNA molecule called crRNA that forms hydrogen bonds with a specific target DNA sequence. The crRNA is covalently linked to a stem-loop RNA sequence call tracrRNA. |
| What does tracrRNA do? | The tracrRNA binds to and activates the Cas9 endonuclease to cut the double-stranded DNA at the target sit. |
| Describe the second CRISPR-Cas9 molecular component. | A CRISPR-associated endonuclease protein(Cas) is a non-specific endonuclease that cuts double stranded DNA when activated by the tracrRNA. The genome editing system described below uses the Cas9 enzyme isolated from the bacterium streptococcus pyogenes |
| What does the DNA sequence targeted by sgRNA need to contain? | The DNA sequence targeted by the sgRNA needs to contain a protospacer adjacent motif (PAM) sequence, as Cas9 binds to the PAM sequence to position itself while it cuts both strands of the DNA. |
| What is the PAM sequence in CRISPR? | The PAM sequence is a DNA consensus sequence consisting of 5'-NGG-3' where N is any of the DNA bases(A,T, C, or G)The PAM sequence is in the nontarget DNA strand; nontarget DNA strand does not form hydrogen bonds the the crRNA component within the sgRNA |
| What is the PAM in the nontarget DNA strand? | The PAM in the nontarget DNA strand is located 3-4 nucleotide in the 3' direction (downstream) from the site that will be cut by Cas9. |
| What is the 1st step in the CRISPR-Cas9 systems that creates the genome edits? | Cas9 binds to the PAM sequence in the nontarget DNA strand. |
| What is the 2nd step in the CRISPR-Cas9 systems that creates the genome edits? | The target and nontarget DNA strands are separated from each other. The Cas9 enzyme is the helicase that separates the two DNA strands. |
| What is the 3rd step in the CRISPR-Cas9 systems that creates the genome edits? | crRNA attempts to form hydrogen bonds with target DNA strand. If crRNA forms proper hydrogen bonds with target DNA strand, then genome editing continues. |
| What happens in the 3rd step if the hydrogen bonds fail to form? | If hydrogen bonds fail to form Cas9 enzyme is released and binds to another PAM sequence in the genome. |
| What is the 4th step in the CRISPR-Cas9 systems that creates the genome edits? | The binding of the crRNA to the target DNA strand activates tracrRNA, which in turn, activates Cas9 |
| What is the 5th step in the CRISPR-Cas9 systems that creates the genome edits? | Cas9 enzyme cuts both DNA strands 3-4 nucleotides in the 5' direction (along the nontarget strand) from the PAM site. |
| What is the 6th step in the CRISPR-Cas9 systems that creates the genome edits? | Once both strands of the DNA have been cut by Cas9 the cells DNA repair system attempt to fix the break in the dsDNA and in doing so, add a few bases, delete a few bases, or insert a completely new piece of DNA. |
| What is the natural function of CRISPR-Cas9? | The CRISPR-Cas9 system is thought to be analogous to an immune system, protecting bacteria against invading bacteriophages (virus that infect bacteria). During an infection, the bacteriophage genome is injected into the cytoplasm of the bacterial cell |
| What happens to the bacteriophage in the natural function of CRSPR-Cas9? | The bacteriophage DNA is cut by nucleases, and a portion of the bacteriophage genome is stored in the CRISPR gene locus. |
| What is the CRISPR gene locus? | Overall, CRISPR gene locus in bacteria consists of clusters of repetitive DNA sequences (short palindromic repeats that are 30-40 base pairs in length) separated by bacteriophage DNA sequences called spacers. |
| What is the spacer sequence in the CRISPR? | In essence the spacer sequences within the CRISPR locus are a library of previous bacteriophage infections |
| What happens to the CRISP gene locus? | Upon reinfection with the same bacteriophage, the CRISPR gene locus is transcribed to produce two types of RNA molecules. |
| What happens with the spacer sequence in CRISPR? | The spacer DNA sequence (the bacteriophage genome) is transcribed to produce the single-stranded CRISPR RNA (crRNA) to form hydrogen bonds with the DNA of the infecting bacteriophage. |
| What happens with another gene in the CRISPR locus? | Another gene in the CRISPR locus is transcribed to make the transactivating crRNA (tracrRNA). The crRNA and the tracrRNA form hydrogen bonds with each other and then bind to the Cas 9 endonuclease. |
| What is something to note in the other gene in the CRISPR locus? | Note: that the tracrRNA contains the stem-loop that activates Cas9. The crRNA:tracrRNA:Cas9 complex then binds to a PAM sequence in the DNA of the invading bacteriophage. |
| What is something to note in the other gene in the CRISPR locus? Part 2 | The two DNA strands within the bacteriophage DNA are separated and the crRNA forms hydrogen bonds with the target DNA strand, while the nontarget DNA strand is moved out of the way. |
| What is the final thing about the natural function of CRISPR-Cas9 | Finally, the Cas9 protein makes double-stranded breaks (DSB) in the DNA of the bacteriophage, thereby destroying the bacteriophage genome and inhibiting the bacteriophage infection. |
| Application of CRISPR-Cas9. | Genome editing via CRISPR-Cas9 involves designing a 20 nucleotide long crRNA sequence that forms hydrogen bonds with a target DNA sequence of interest. This crRNA is covalently linked to the tracrRNA that forms the RNA stem-loop to active Cas9. |
| What is the sgRNA component of CRISPR-Cas9 system? | The crRNA linked to the tracrRNA is the sgRNA component of the CRISPR-Cas9 system. |
| Are sgRNA and Cas9 DNA sequences ligated in seperate cloning sites? | Both the sgRNA and Cas9 DNA sequences are ligated into separate cloning sites within a plasmid vector, and the plasmid vector is introduced into a cell of interest, including a eukaryotic cell. |
| What happens with transcription of cloned genes? | Transcription of the cloned genes lead to the production of both the sgRNA and Cas9 molecules. |
| What happens when you bind the sgRNA to a target DNA sequence? | When the sgRNA binds to a target DNA sequence, Cas9 produces a double stranded break (DSB) in the DNA. When the cell attempts to repair these DSBs the cell can undergo the non-homologous end joining (NHEJ) DNA repair pathway. |
| NHEJ? | NHEJ is not perfect and insertion or deletion of a few nucleotide occurs (these mutations are called indels) as the DSB is repaired. |
| What do indels do? | Recall that indels cause a frameshift during translation that ultimately prevent the eukaryotic gene from making a functional protein product. |
| What is the results of using CRISPR-Cas9 genome editing followed by NHEJ? | Therefore, CRISPR-Cas9 genome followed by NHEJ allows the researcher to produce a gene knock-out cell line or organisms. The knockout fails to produce a functional protein product. |
| What is the double strand break in DNA that can also lead to another type of DNA repair? | Double strand breaks in the DNA can also lead to another type of DNA repair known as homology directed repair (HDR). |
| What does DNA repair allow? | In this case, DNA repair allows the insertion of a donor sequence at the location of the DSB, instead of repairing the break by inserting or deleting a few nucleotides. The donor DNA can be engineered to contain a mutant form of a gene. |
| How does the approach in a donor of a mutant gene happen? | This approach allows the researcher to insert a mutant form of a gene. This approach allows the researcher to insert a mutant gene in the place of a wild-type gene to study the effects of the mutation on the cell. |
| Wat can a donor DNA sequence contain? | Alternatively, the donor DNA sequence can contain a wild-type version of a gene that replaces the mutant form of the gene within the cell. The replacement of a gene with a different allele of the same gene produces a knock-in cell. |
| Why is CRISPR-Cas9 a convenient genome editing system to use? | CRISPR-Cas 9 is a convenient genome editing system to use because if a scientis wishes to study a different gene, the scientist designs a new 20-nucleotide-long crRNA that forms hydrogen bonds with the new target gene, |
| Why is CRISPR-Cas9 a convenient genome editing system to use? part 2 | all of the other components (tracrRNA, Cas9) of the CRISPR-Cas9 system remain the same. |
| Challenge associated with CRISPR-Cas9 | In a typical experiment, the research will introduce the CRISPR-Cas9 vector into a population of eukaryotic cells. Because the process of genome editing is inefficient the experiment will result in three groups of cell in the population. |
| What three groups of cells in a population will happen because the process of genome editing is inefficient? | Those is which no editing occurred, those in which one of the two alleles of a gene is edited, and those with both alleles edited. If the knock-out approach is used the researcher will want to study cells that have no functional copies of the gene. |
| What is the end of if a scientists do not have a functional copy of the gene? | Therefore the researcher will need to identify those cells with both alleles edited. Determining the DNA sequence of the target gene in individual sells is one of the easiest ways to confirm that the desired changes have taken place. |
| What is the crRNA designed to do? | The crRNA is designed to target a specific gene in the genome; however sometimes a 20 nucleotide-long crRNA can bind to more than one DNA sequence in the genome simultaneously. |
| What does the crRNA raise the possibility of? | This raises the possibility that the CRISPR-Cas9 system will cut the DNA at undesired locations within the genome producing off-target effects. |
| What is a bad thing of producing off target effects? | Because the locations of these off-target cut sites are difficult to predict, treating cells with CRISPR-Cas9 can have unintended consequences on the cell or organism. |
| The ethics of Genome Editing! | Many scientists are interested in using CRISPR-Cas9 to treat human genetic diseases, especially diseases for which there is currently no treatment. There are two main ways that human genome editing can be used to treat disease. |
| What are the two ways genome editing can be used to treat disease? | Inactivation of a mutant gene to remove its effects on the cell (using the NHEJ knock-out approach) or insertion of a functional allele to replace a mutant one (using the HDR knock-in approach). |
| What is uncertain about the ethics of using CRISPR-Cas9? | With the promise that CRISPR-Cas9 brings there is also uncertainty about the ethics of this technique, particularly when applied to humans. |
| What do researchers think about CRISPR-Cas9? | Most researchers agree that if we have the tools to necessary to treat a genetic disease we should use those tools to improve the lives of patients. |
| What is the disagreement about the CRISPR-Cas9? | However, considerable disagreement exists as to whether the CRISPR-Cas9 technique should be used to modify the germ-line cells that produce gametes or embryos. |
| What is permitted with research for CRIPR-Cas9? | Research on human embryos is permitted if the treated human embryos are destroyed day 14 of development and are not implanted into the womb. |
| What is a important issue to consider for genome editing? | An important issue to consider with genome editing is that of informed consent. |
| What is a adult gives consent? | An adult can give consent for genome editing that can potentially treat their genetic disease, but when the treatment extends to future there is no way to obtain consent (the developing fetus cannot give consent. |
| What is the public opinion about genome editing? | Public opinion remains divided as to who has the right to make the decisions for the fetus; is it the person who develops from the embryo, parents, or the government. |
| What happened in November 2018? | In November 2018 the press announced that a researcher in China used CRISPR-Cas9 to successfully edit the CCR5 gene in human twins (one received the edit while the other did not) |
| What happens if you knock out the CCR5 gene in human twins? | Knocking out this gene is expected to prevent the treated child from contracting a human immunodeficiency virus (HIV) infection. To say the scientific world was upset about this announcement is an understatement. |
| What happened in November 2018 with a child? | This was the first time that a human baby was born after genome editing was performed. |
| Why was the announcement not receive with congratulations? | The reason why this announcement was not received with congratulations was, in part due to the lack of informed consent and the failure to make sure that no off-effects took place before implanting the embryos into the birth mother. |
| What was the reason people did not believe the parents were informed? | In fact, is is uncertain if the parents were informed as to the genome editing experiment or if they were coerced into giving their consent. |
| What is an international agreement that can be made about genetics? | There may never be an international agreement concerning genome editing that can be enforced by all nations. |
| Why may there never be a international agreement concerning genome editing? | Even when there is agreement as to what is ethical and what is not, there will always be individuals or nations who will carry out research that is contrary to the moral beliefs of others. |
| What are some important questions to consider about genome editing? | Important questions to consider include how do we establish laws concerning the ethical practice of scientific research and how do we penalize those who knowingly disobey those laws? |
| What happened in 1956? | In 1956 mathematician and biologist Jacob Bronowski wrote that as scientists, We ought to act in such a way that what is true can be verified to be so. An expression of his belief that it is our right and our duty to explore the unknown and seek truth. |
| What is the point about international committees? | The point is that maybe having large international committees decide what should be practiced and what should be prohibited is not the real question, |
| What is the real question international committees should be asking? | but rather how can society ensure that research done is based on the principle of seeking truth to better the lives of humankind? |
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