LECTURE 11 - GENOME EDITING Flashcards
Definition of genome editing:
To induce changes to the sequence of genomic DNA in a controllable manner.
‘→In living cells, efficiently and permanently’
History of genome editing 4
- Designer DNA binding proteins
→Zinc Finger Proteins
→TALENS - Zinc finger protein
designed to bind to this
specific DNA sequence - ENDONUCLEASE DOMAIN CUTS
- PROBLEM: A new protein needs to be designed and made for each new DNA target site = $$ and time
CRISPR:
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas9 genome editing…PROCESS?
- → Evolved as a way of prokaryotes protecting themselves from viruses
- → Cas9 endonuclease protein is directed to a specific DNA site by a short
‘RNA’ guide that is loaded into the protein
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats …ADVANTAGES? = 3
- Much greater efficiency of targeting genomic loci
- ease of designing, constructing and delivering multiple sgRNAs, allowing
multiplexed genome editing in mammals. - Each new target site only
requires a new guide RNA to be designed, not a whole new protein
Cas9-guide-target DNA structure…DIAGRAMS
SLIDE 6
- Schematic represenation of cas9, sgRNA and target DNA during the recognition step of genome editing, proir to cleavage
- Structural basis of the interaction as detailed in the crystal structure (4un3)
Development of the CRISPR-Cas9 system:
ORIGINAL
….3 COMPONENTS TO BE IMPORTED:
three components to be imported:
- guideRNA
- tracrRNA
- Cas9
Development of the CRISPR-Cas9 system:
ORIGINAL DIAGRAM
SLIDE 7
Development of the CRISPR-Cas9 system:
UPGRADED
2 COMPONENTS
two components to be imported
- Chimeric guide sgRNA
- Cas9
Development of the CRISPR-Cas9 system:
UPGRADED
DIAGRAM
SLIDE 8
How does the cell repair the cut DNA?
Two main DNA repair mechanisms in cells:
- Non-homologous end joining
- homologous recombination
How does the cell repair the cut DNA?
Two main DNA repair mechanisms in cells: 4
NON HOMOLOGOUS END JOINING
- process
- advantages/disadvantages
- ligation
2.Can result in insertions and deletions of small numbers of bases into the DNA at the cut site
2 * Error-prone
3 * Faster
4 * Commonly used mechanism
How does the cell repair the cut DNA?
Two main DNA repair mechanisms in cells: 6
HOMOLOGOUS RECOMBINATION
- ‘sister’ chromatin used as template for
missing DNA sequence
…..2. EXCISON FILLIN EXCISION
3 * Low error rate
4 * Slower
5 * Less commonly used
6 * Cell needs to be in S phase
How does the cell repair the cut DNA?
Two main DNA repair mechanisms in cells: DIAGRAM
SLIDE 9
Applications of genome editing …
- Add new sequence into DNA
- Modify existing sequence (eg. Point mutation)
- Delete DNA
- Inactivate a protein-coding gene
Applications of genome editing…
1. Add new sequence into DNA
EXPLAIN
→harness the ‘homologous recombination’ mode of DNA
repair (also called ‘homology directed repair’ HDR)
- Artificially introduced DNA used as template for repair
DIAGRAM SLIDE 10
Case study: Editing the genome of cell lines…
- SFPQ (splicing factor proline and glutamine rich) is a transcription factor found in subnuclear bodies called paraspeckles
- Overexpression of SFPQ and variants is problematic
- Use CRISPR-Cas9 to engineer the genome to create endogenous GFPSFPQ
Case study: Editing the genome of cell lines DIAGRAM
SLIDE 11
Applications of genome editing
- Modify existing sequence (eg. Point mutation)
EXPLAIN
- cut
- EXCISON
- Repair with introduced template by homologous recombination
OR
Use a ‘base editor’
DIAGRAM SLIDE 12
Applications of genome editing
3. Delete DNA
EXPLAIN
→harness the ‘non homologous end joining’ mode of DNA repair
- 2 cuts
- DNA repair by NHEJ
DIAGRAM ON SLIDE 13
Applications of genome editing
4. Inactivate a protein-coding gene
EXPLAIN
- cut
- excision
- Repair with non homologous end joining with errors
Indel (insertion/deletion)
will disrupt the protein
coding frame of the gene
(frameshift mutation)
DIAGRAM ON SLIDE 14
Application: dCas9 (dead cas9) EXPLAIN
- dCas9: A mutant Cas9 protein, that cannot cut DNA
- Useful for targeting fused transcriptional activation, or repression domains, to a specific DNA sequence, but without cutting/editing the
DNA
Application: dCas9 (dead cas9)…
main uses for dcas9
DIAGRAM
SLIDE 15
Application: dCas9 (dead cas9)
Specific transcriptional activation domains can be fused to dcas9: DIAGRAM
SLIDE 15
Limitations of CRISPR-Cas9 = 3
- Cas9 can cut other, unwanted sites in the genome (‘off-targets’)
- Choice of cutting site is limited by need for the PAM (NGG) site
- Cas9 is a large protein, therefore gene encoding it not easy to be packaged into a virus for delivery
Solutions to CRISPR-cas9 limitations =
- CRISPR/Cas 9 double nickase system to reduce off targets
- High-fidelity CRISPR–Cas9 nucleases with no detectable genomewide off-target effects. Kleinstiver et al 2016. Nature.
- 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’
Solutions to CRISPR-cas9 limitations DIAGRAM
SLIDE 17
Base editors (genome engineering without cutting the dsDNA)… WHAT IS IT? WHY?
- 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).
- →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
Base editors (genome engineering without cutting the dsDNA) DIAGRAM
SLIDE 18..LABELLED
Base editors (genome engineering without cutting the dsDNA)… PROCESS = 4
- The fused base modification enzymes modify single-stranded DNA (ssDNA), but not double stranded DNA (dsDNA).
- Base pairing between the gRNA and target DNA forms a small segment of single stranded DNA in an “R-loop”.
- DNA bases within this ssDNA bubble are modified by the deaminase enzyme.
- 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.
Base editors (genome engineering without cutting the dsDNA) PROCESS DIAGRAM
SLIDE 19
BIOTECH applications = 4
- Agriculture
→Insert genes or modify genes to create desirable traits - Molecular cell biology
→Modify genomes of human cell lines for biomedical research - Animal models
→Much faster way of creating transgenics, disease models, knockout mice - Gene Drives
→Used for population control of pests eg. mosquitoes
Aside: Injections into mouse zygotes
(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)
Gene drives…(EXPLAIN) = 4
Insert genes encoding CRISPR machinery and
accompanying ‘payload’ gene into DNA.
2.CRISPR will be activated in zygote to cut sister chromatin.
- HR will be used
to insert CRISPR sequence into sister chromatin. - Results in nonmendelian
inheritance of the CRISPR machinery
CLINICAL applications
- CELL THERAPY
- GENE THERAPY
CLINICAL applications - CELL THERAPY = 4
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
CLINICAL applications: GENE THERAPY = 3
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
Understanding human germline editing… 2
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
Approved work using CRISPR-Cas9 in viable human embryos = 2
- 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.
Human germline editing…legal regulation = 3
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
Human germline editing PROBLEMS = 3
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
Ethical debate = 3
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.
SUMMARY = 5
- Genome editing involves cutting the DNA, then harnessing the DNA repair mechanisms of the cell to enact a change
- 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
- CRISPR Genome editing can be used to add DNA, delete DNA, and make single point mutations in DNA
- Applications in clinical use and biotechnology
- Controversial: possible future human genome editing that can be passed down to the next generation
