Genome Editing
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| Genome Editing | show 🗑
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| What is the goal of genome editing? | show 🗑
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| What is the application of genome editing? | show 🗑
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| show | 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.
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| show | Finally genome editing can be used to enhance the yield of crops or give desirable traits to livestock.
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| How does genome editing work (1) | show 🗑
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| show | 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.
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| show | 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.
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| show | Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9.
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| show | 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.
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| What happens in the ZFN process when the endonucleases cut? | show 🗑
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| show | 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.
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| show | 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.
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| How is a CRISPR-Cas9 used? | show 🗑
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| show | 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.
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| show | 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.
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| show | It is currently used all over the world and has a promising future.
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| show | CRISP is an acronym for the clustered regularly interspaced short palindromic repeats (CRISPR) system. It has two molecular components.
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| show | 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.
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| What does tracrRNA do? | show 🗑
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| Describe the second CRISPR-Cas9 molecular component. | show 🗑
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| show | 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.
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| What is the PAM sequence in CRISPR? | show 🗑
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| What is the PAM in the nontarget DNA strand? | show 🗑
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| What is the 1st step in the CRISPR-Cas9 systems that creates the genome edits? | show 🗑
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| What is the 2nd step in the CRISPR-Cas9 systems that creates the genome edits? | show 🗑
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| show | crRNA attempts to form hydrogen bonds with target DNA strand. If crRNA forms proper hydrogen bonds with target DNA strand, then genome editing continues.
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| What happens in the 3rd step if the hydrogen bonds fail to form? | show 🗑
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| What is the 4th step in the CRISPR-Cas9 systems that creates the genome edits? | show 🗑
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| What is the 5th step in the CRISPR-Cas9 systems that creates the genome edits? | show 🗑
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| What is the 6th step in the CRISPR-Cas9 systems that creates the genome edits? | show 🗑
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| show | 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
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| show | The bacteriophage DNA is cut by nucleases, and a portion of the bacteriophage genome is stored in the CRISPR gene locus.
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| What is the CRISPR gene locus? | show 🗑
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| show | In essence the spacer sequences within the CRISPR locus are a library of previous bacteriophage infections
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| show | Upon reinfection with the same bacteriophage, the CRISPR gene locus is transcribed to produce two types of RNA molecules.
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| show | 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.
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| What happens with another gene in the CRISPR locus? | show 🗑
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| show | 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.
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| show | 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.
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| What is the final thing about the natural function of CRISPR-Cas9 | show 🗑
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| Application of CRISPR-Cas9. | show 🗑
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| What is the sgRNA component of CRISPR-Cas9 system? | show 🗑
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| Are sgRNA and Cas9 DNA sequences ligated in seperate cloning sites? | show 🗑
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| show | Transcription of the cloned genes lead to the production of both the sgRNA and Cas9 molecules.
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| What happens when you bind the sgRNA to a target DNA sequence? | show 🗑
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| NHEJ? | show 🗑
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| What do indels do? | show 🗑
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| What is the results of using CRISPR-Cas9 genome editing followed by NHEJ? | show 🗑
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| What is the double strand break in DNA that can also lead to another type of DNA repair? | show 🗑
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| show | 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.
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| How does the approach in a donor of a mutant gene happen? | show 🗑
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| show | 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.
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| show | 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,
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| Why is CRISPR-Cas9 a convenient genome editing system to use? part 2 | show 🗑
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| show | 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.
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| What three groups of cells in a population will happen because the process of genome editing is inefficient? | show 🗑
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| show | 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.
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| show | 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.
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| show | This raises the possibility that the CRISPR-Cas9 system will cut the DNA at undesired locations within the genome producing off-target effects.
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| show | 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.
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| show | 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.
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| What are the two ways genome editing can be used to treat disease? | show 🗑
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| What is uncertain about the ethics of using CRISPR-Cas9? | show 🗑
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| What do researchers think about CRISPR-Cas9? | show 🗑
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| show | 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.
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| show | Research on human embryos is permitted if the treated human embryos are destroyed day 14 of development and are not implanted into the womb.
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| What is a important issue to consider for genome editing? | show 🗑
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| show | 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.
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| show | 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.
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| What happened in November 2018? | show 🗑
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| show | 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.
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| show | This was the first time that a human baby was born after genome editing was performed.
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| Why was the announcement not receive with congratulations? | show 🗑
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| What was the reason people did not believe the parents were informed? | show 🗑
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| show | There may never be an international agreement concerning genome editing that can be enforced by all nations.
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| show | 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.
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| What are some important questions to consider about genome editing? | show 🗑
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| show | 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.
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| What is the point about international committees? | show 🗑
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| What is the real question international committees should be asking? | show 🗑
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