12.2/12.3 Flashcards
what is CRISPR-Cas?
- designed to target specific DNA molecules, comparable to adaptive immune system of vertebrates
- found in 50% of bacteria, 90% of archaea
What does CRISPR stand for? How about CAS?
CRISPR: Clustered regularly spaced palindromic repeats
CAS: crispr associated repeats
What are the 3 steps of CRISPR?
-1. spacer acquisition
- 2. expression of cRNAs
3. interference (performed with the effector complex)
What are the 3 components of the effector complex?
- cas 9 enzyme
- cRNA
- tracrRNA: transactivating crispr RNA
- (guide strand is a combo of the last two)
what is the protospacer adjacent motif? (PAM)
- NGG (N=any nucletodie) next to space sequence (tells it to check for match)
- not found in any CRISPR array
- simple and common elsewhere
Who discovered the use of CRISPR?
- Doudner/charpentier
what is the key innovation of CRIPSR?
- substitution of chimeric gRNA in place of natural cRNA and tracRNA
describe the process of editing the genome with CRISPR-Cas
- sgRNA is designed to target a specific sequence in genome
- sgRNA complex assembles with the CAS-9 protein to form the effector complex
- them effector complex finds a PAM and cas9 unwinds DNA immediately upstream
- if target sequin present, the 20bp 5’ end of gRNA binds with it, cas9 makes a double stranded cut in the genome
What are the two possible methods of DNA repair?
- 1) NHEJ: broken ends can be rejoined without any template strand (ie: duplication or deletion) - Non homologous chromosomes joining
2) HDR: broken ends can be rejoined using a template (donor DNA)
- homology directs repair
Describe NHEJ. What occurs if there are no INDELs?
- non homologous end joining
- most common type of repair to repair a double strand DNA break
- no template used!!!
- instead nucleotides may be inserted or deleted as the ends are rejoined (results in INDELS)
No INDELS: CAS9 keeps cutting the site until a mutation does occur: resulting frameshift leads to non-functional gene (gene ‘knockout’)
Describe HDR.
- Homology Directed Repair
- uses same repair enzymes as in crossing over
- can use homologous chromosomes as template
- can inject donor DNA to stimulate HDR
What are some advantages of CRISPR-Cas9?
- relatively cheap and easy
- targeting: can design sgRNA to target any sequence desired
- relatively specific
- gene knockouts (NHEJ) can be used to silence and see the impacts of a gene
- can be introduced to living cells
- can introduce CAS9 with donor to stimulate HDR
What are some challenges of CRISPR?
- off target effects: cleavage sometimes no specific
- modified CAS9 has been created to use longer target sequence, but is slower
- can be hard to control whether NHEJ or HDR is used
- germ line cells have advanced HDR
- Mosaicism: delivery of CAS9 not 100% for all cells, challenge for multicellular organisms
what is mosaicism? why is it a challenge for CRISPR? What is a potential solution?
- not 100% of the cells have CAS9 delivered: a challenge for multicellular organisms
- solution: injection CAS9 at the single cell stage
What are some methods of delivery for CAS9?
- transfection, microinjection, electroporation
how can CRISPR be used for basic research?
- uses gene knockouts to determine unknown gene functions
- sometimes knocking out a gene results in a desirable phenotype
What are some examples of ‘hacking’ genomes to meet human needs?
- animal organs
- de-extinction of species
reversing mutations
eliminate insect spread diseases
domestication of new plants for agriculture
improved farm animals
Describe genome edited pigs and virus
- designer live stock
- PPRS virus kills pigs: researchers used CRISPR to cut out a part of the surface protein coded by CD163 needed by the virus: result are pigs that are not impacted by the virus
(protein receptor remained functional, but PPRS viruses were blocked from entry)
Describe genome edited pigs and their diet
- Bguconase, xylonasae, phase break down matter pigs don’t digest: inserting genes that produced these enzymes in pigs salivary glands
Describe CRISPRs use in tomatoes
- knocking out 2 genes made tomatoes 30% sweeter: no DNA added so theoretically easier for market approval
Describe CRISPRs use in bananas
- fungal disease threatens banana: because of their sterility it is difficult or impossible to breed a resistant banana
- CRISPR now being used to improve resistance of bananas to disease/pests and improve nutritional qualities
how can CRISPR be used to tackle sickle cell anemia?
- mutation causes RBCs to sickle
- blood stem cells removed, culture, gene that codes for off switch is knocked out with CRISPR using NHEJ
- edited cells reintroduced to patient
what diseases can be treated with CRISPR?
- sickle cell anemia, beta thalassemia, and malaria
what is a gene drive?
- a DNA constrict gains gRNA sequence, CAS9, payload gene, flanking sequences
- once introduced as one copy iy copies itself to homologous chrosome using HDR : sexual reproduction offspring are converted to homozygous
- the payload gene can spread rapidly through population
- could be used insert gene for resistance of malaria or that reduces fertility of mosquito
What is gene drive inheritance?
- the altered gene is always inherited
what are the steps of the mammoth steppe?
- find preserved wooly mammoth DNA
- sequence the genome
- sequence asian elephant genome
- identify cold weather genes
- derive cells, prepare multiplex edit designs (testing multiple genes at a time)
- insert gene edits and create cell line
- test gene edits
- nuclear transfer and fertilization
- implant embryo into surrogate = birth
What parts did scientists combine to form the transgenic Atlantic salmon?
- combined growth hormone from chinook salmon with promoter and terminator sequence for antifreeze gene from ocean pout
- this was in order to ensure that the growth hormone was strongly expressed (they didn’t actually want the antifreeze, only the ‘on switch’)
- gene constrict inserted directly into salmon eggs
How are the combined genes expressed in the transgenic salmon?
- the growth hormones are relatively similar (95% aa, 98% aa similarity)
- Ocean pout antifreeze promoter shown to be constitutively active in Atlantic salmon as opposed to normal salmon which only have it in response to environmental cues
what occurred after injecting the gene construct into the salmon egg?
- further breeding showed that the constrict was stable integrated into the Atlantic salmon genome: creation of strain that constitutively expressed chinook salmon GH
- the gene construct was shown to have rearranged itself where part of the promoter moved downstream but the fragmented promoter still worked but at a slightly lower level
- aqua advantaged salmon trademarked!
What are some steps taken to reduce the risk of interbreeding between transgenic and wild salmon?
- triploid females sterile: pressure treated eggs
- production of neomales: sex reversed females by methyl testosterone treatment
- salmon growth in closed systems on land rather than in net pens: cost effective since salmon grow so much faster
How is rhizobium radiobacter coopted to naturally introduce new genes to plants?
1) natural gene transfer: the agrobacteirum invades plant cell at a wound
2) part of the Ti plasmid transferred to the plant cell
- 3) integrates into one of the plant chromosomes
this method can be used to introduce foreign DNA into plants
what is the general approach to introduce foreign DNA into plants?
1) foreign DNA is inserted into a plasmid vector
2) transferred to agrobacterium with a Ti plasmid
3) the helped Ti plasmid is required for infection
4) the plasmid vector along with Andy foreign DNA, is transferred to a plant cell where it integrates into a plant chromosome
Why is Bacillus thuringiensis a desirable insertion into plant chromosomes?
- it produces BT toxin which is lethal to many pests but not to humans or other animals, biodegradable
- BT gene has been transferred to many plants using R. Radiobacter
What proportion of BT is ideal for pest resistance?
- most plants had ~2/3 Bt gene inserted, since the full length gene was not effective
- Bt gene was stably integrated into genome, similar approach used for other plant species
What is often repurposed as molecular DNA scissors?
- restriction endonuclease
what method should be used to create modest amounts, or significant amounts, of DNA?
PCR is useful for modest amounts of DNA but when large amounts are needed cloning is best
what is used to check for the success of experiments?
PCR is used to check for success
- gel electrophoresis, in combination with PCR or RE cleavage, is used to check for success of cloning/transformation experiments
Describe the process of inserting BT genes to plants
- cloned BT is inserted into e.coli gene for replication
- restriction enzymes used to produce varying lengths of the BT gene: ligated to a Neo gene (has kanamyacin resistance)
- constructs inserted into expression vector (also contains promoter and Poly A consensus sequences for proper expression)
- neo+Bt+ plasmids recombined with Ti plasmids inside the bacterial cell
- whole plants regenerated from plants cells, checked for stability and kanamycin resistance: ~2/3 of the BT gene integrated
