LECTURE 11 - GENOME EDITING Flashcards

Question

Limitations of CRISPR-Cas9 = 3

Answer 1

1. Cas9 can cut other, unwanted sites in the genome (‘off-targets’) 2. * Choice of cutting site is limited by need for the PAM (NGG) site 3. * Cas9 is a large protein, therefore gene encoding it not easy to be packaged into a virus for delivery

Answer 2

1. CRISPR/Cas 9 double nickase system to reduce off targets 2. High-fidelity CRISPR–Cas9 nucleases with no detectable genomewide off-target effects. Kleinstiver et al 2016. Nature. 3. New Cas9-like proteins, smaller, and not dependent on PAM recognition are being sought. - 'smaller size, means more likely to be packaged in a virus for delivery'

Answer 3

1. Base editing is a new approach that uses components from CRISPR systems and other enzymes to directly make point mutations in DNA without making double stranded DNA breaks (DSBs). 2. →Many genetic point mutations that cause disease could be fixed by a single base change (eg. A.T to G.C) 3.dCas9 fused to deaminase enzyme that converts one base to another

Answer 4

SLIDE 18..LABELLED

Answer 5

1. The fused base modification enzymes modify single-stranded DNA (ssDNA), but not double stranded DNA (dsDNA). 2. Base pairing between the gRNA and target DNA forms a small segment of single stranded DNA in an “R-loop”. 3. DNA bases within this ssDNA bubble are modified by the deaminase enzyme. 4. To improve efficiency in eukaryotic cells, the dCas9 also creates a nick in the non-edited DNA strand, inducing cells to repair the non-edited strand using the edited strand as a template.

Answer 6

1. Agriculture →Insert genes or modify genes to create desirable traits 2. Molecular cell biology →Modify genomes of human cell lines for biomedical research 3. Animal models →Much faster way of creating transgenics, disease models, knockout mice 4. Gene Drives →Used for population control of pests eg. mosquitoes

Answer 7

(a) Hold the zygote and cut the zona pellucida using piezo pulses. (b) Expel the zona fragment and cytoplasm from the pipette, and push the injection mix to the tip of the pipette. (c) Insert the injection pipette into the zygote until it almost reaches the opposite side of the zygote's cortex. (d) Push the mix forward again until it forms a droplet outside the oolemma. (e) Apply one weak piezo pulse to puncture the oolemma at the pipette tip. (f–h) Immediately withdraw the injection pipette from the zygote to the zona pellucida and then slowly suck the cytoplasm into the pipette, allowing closure of the zygote's oolemma. All injections are performed with an 20× object lens at room temperature (25–30°C)

Answer 8

Insert genes encoding CRISPR machinery and accompanying ‘payload’ gene into DNA. 2.CRISPR will be activated in zygote to cut sister chromatin. 3. HR will be used to insert CRISPR sequence into sister chromatin. 4. Results in nonmendelian inheritance of the CRISPR machinery

Answer 9

1. CELL THERAPY 2. GENE THERAPY

Answer 10

1 →Isolate patient cells 2 →Edit genome of cells in culture (eg. To correct mutation, or express immunotherapy molecule) 3 →Re-implant cells into patient 4 →Eg. Hematopoietic stem cells to correct b-thalassemia or CAR-T therapy in cancer

Answer 11

1 →Deliver CRISPR Cas9 machinery directly to affected cells/tissues in the patient 2 →More challenging. Currently relies on delivery of CRISPR inside engineered viruses 3 →Eg. Duchenne muscular dystrophy

Answer 12

1 →With CRISPR and IVF (in vitro fertilization) it is now possible to edit the genome of a fertilized human egg. 2.→These DNA changes will be passed to all the cells in the baby, including the ‘germ’ cells that will go on to make future generations

Answer 13

* Kathy Niakan used CRISPR–Cas9 in healthy human embryos to delete OCT4 (Fogarty et al Nature 2017). * Experiments were stopped after seven days (256 cell stage), after which the embryos were destroyed.

Answer 14

1 → Legal regulation framework varies in different countries 2 → 2018: He Jiankui (Guangdong, China) claimed to have created the first human CRISPR genome edited babies (‘Lulu and ‘Nana’). 3 → The rationale was to alter the CCR5 gene to create a HIV resistant mutation

Answer 15

1 * Although their father was HIV positive, it would have been possible with current practice to prevent transmission of HIV 2 * the mutations created in Lula and Nana alter CCR5 in ways not seen before 3 * Parents were apparently informed and consented, but original hospital ethics applications are suspect

Answer 16

1 * Several summits of scientists declare CRISPR should not be used to modify human embryos that are intended for use in establishing a pregnancy. 2 * Public opposition to embryo research could cause a backlash against use of genome editing as therapy. 3 * A complete ban on embryo research is unrealistic: even if some researchers agree to abstain from editing embryos, or if some countries ban it outright, such work will inevitably be done in places with less oversight.

Answer 17

1. Genome editing involves cutting the DNA, then harnessing the DNA repair mechanisms of the cell to enact a change 2. The key technology breakthrough is that the CRISPR system requires an RNA guide, rather than a designer protein, to direct Cas9 to a DNA target 3. CRISPR Genome editing can be used to add DNA, delete DNA, and make single point mutations in DNA 4. Applications in clinical use and biotechnology 5. Controversial: possible future human genome editing that can be passed down to the next generation

LECTURE 11 - GENOME EDITING Flashcards

(43 cards)