CRISPR and biotech Flashcards
(22 cards)
what is the CRISPR-Cas 9?
- clustered regularly interspersed short palindromic repeats-CRISPR-associated protein 9
- defence mechanism against invasion of bacteriophages
Describe the components of the CRISPR locus
- Cas genes
structural genes that code for endonucleases (cas proteins) - Repeat-spacer array / CRISPR array
repeats (short palindromic DNA sequences, all the same)
interspersed with spacers (unique DNA sequences from previous bacteriophage infections)
acts as a memory bank of past viral infections
repeats act as a scaffold, allowing spacers to come in
What are PAMs?
- NGG
- found next to CRISPR targets in bacterial DNA
- not found in bacterial CRISPR array
- ensure bacterias own genome is not damaged by Cas enzymes
(if there’s no Pam site, may have to use different technique)
Outline Spacer Acquisition
- Bacteriophage invades bacterial cell, injecting its DNA into cell
- Cas 1 and Cas 2 capture pieces of injected phage DNA
- Cas 1 and Cas 2 cut phage DNA upstream of PAM site, creating protospacer
- protospacer inserted into CRISPR array as a spacer, new repeat region built after
spacer is inserted into 5’ end of coding strand of CRISPR array
5’ —–coding——–3’
3’——template——5’
Outline CRISPR RNA biogenesis
- CRISPR array transcribed into pre-crRNA
- Cas proteins cleave long RNA molecule into short segments, containing one spacer and parts of repeats. this forms crRNAs
- crRNAs combine with tracrRNA to form guide rna or cr:tracrRNA?
precrRNA: spacer, repeats
tracrRNA (transcribed from other gene)
whole thing: cr:tracrRNA (not crRNA??)
Outline interference
- Cas 9 binds to crRNA, forming the surveillance complex
- searches phage’s genome for sequence matching crRNA spacer. target sequence is called protospacer
- surveillance complex unzips phage DNA to see if crRNA can base-pair with one strand
- if protospacer is complementary to crRNA, cas 9 cleaves phage DNA upstream of PAM to destroy it.
define single guide rna sgRNA
- made in laboratory
- serves same function as crRNA (cr:tracrRNA)
- type of gRNA
Step 1 of CRISPR in the lab: designing sgRNA
- researchers design sgRNA to be complementary to the target sequence in the genome
- add cas9 and sgRNA to the cells they want to edit
- sgRNA is complementary to template strand
Step 2 of CRISPR in the lab: Identifying target DNA
- cas9 carries a sgRNA molecule
- cas9-sgRNA complex searches for matching DNA sequences in a genome, then identifies PAM sequence
- Binds to PAM, then unwinds double helix
- if the sgRNA matches the target upstream of PAM, it will base pair with the complementary strand
Step 3 of CRISPR in the lab: Cutting
- if cas9 finds a PAM next to a region of DNA that matches its sgRNA, cas9 cuts both strands of DNA creating blunt ends
Step 4 of CRISPR in the lab: Genome editing
cellular repair proteins mend the gap and edit the genome at the same time by:
1. stitching the two broken ends back together
- can result in small DNA deletions or insertions as the repair site
- these can inactivate genes
- fix DNA breaks by adding in new DNA sequences
- researches specify the specific DNA sequence
- can fix broken genes or change cellular functions
Applications of CRISPR: photosynthetic efficiency and crop yield
Photosynthesis efficiency:
Aim:
to maximise production of glucose, used for plant growth, increases increases crop yield
e.g improving efficiency of rubisco by reducing its ability to bind to oxygen and undergo photorespiration
Crop yield:
- protect and secure crop yields for a growing worldwide population
- CRISPR cas 9 can be used to target certain genes that impact crop yield (by inserting genes to improve yield or knocking out genes that have a negative effect)
Applications of CRISPR: improving crop quality
CRISPR-cas9 can be used to alter gluten and nutritional content, storage quality and visual appearance of crops
Applications of CRISPR: biotic and abiotic stress resistance
- main factors that affect crop yield and quality
biotic:
- resistance to bacterial, fungal, viral diseases or pests
- ensures the crop can be used with greater efficiency and ensures stable world food supply
abiotic:
- temperature, light, soil, air (e.g drought tolerance)
Applications of CRISPR: hybrid breeding
- enables creation of offspring with the desired characteristics from two different breeds
- allows scientists to shorten the growth time for a plant, enabling it to reach maturity earlier, increasing crop yields in the long term
(edits parent plants before they are crossed)
What are microbes
- microscopic organisms (e.g. yeast)
- vital in breakdown and fermentation of biomass
What is biomass?
- organic (carbon based) material from plants or animals
- renewable source of energy
- high energy substance (contains stored energy from sun and photosynthesis- glucose)
e.g wood, crops, garbage, alcohol fuels, landfill gasses - when burned, biomass releases heat which generates electricity
What is biofuel?
- fuel derived from biomass
- e.g. biogas, bioethanol, biodiesel
- renewable
- more carbon neutral
- biomass can be converted to liquid biofuels (like ehtanol, biodiesel) through fermentation by microbes (e.g yeast)
Anaerobic fermentation (biofuel)
- converts biomass into biogas in the absence of oxygen
- harvest sugar from crops like sugar cane
- combined with yeast
- fermented into ethanol
- ethanol is distilled to obtain a higher concentration of alcohol
- bioethanol is used as fuel
- as it is burned, it releases carbon dioxide which is then absorbed by the crops
(byproducts from fermentation can be used as animal feed)
- biomass is also used to deal with waste!
pros and cons of different fermentation substrates
- forest and industrial residues
pros:
substrates that can be fermented directly by yeast (sugary) have the cheaptest pre-treatment
cons:
starchy substrates, dry plant matter (wood), household waste require expensive pre-treatment to break them down into fermentable substrates - agricultural waste
pros:
- cost effective to obtain
- renewable
- abundant
cons:
- transportation can be difficult
- pre treatment to break down biomass can be expensive
conditions that must be met for successful bioethanol production
- optimum temperature
- substrate concentration
- no oxygen
- yeast
