Gene Editing Flashcards
What are 3 relatively new methods of gene editing?
ZFNs
TALENs
CRISPR/Cas9
ZFNs and TALENs came before CRISPR
What do those 3 gene editing tools have in common?
They are sequence specific nucleuses that cause double strand breaks in the DNA and can either lead to NHEJ or homology-directed repair (HDR) when a template is introduced
Describe what ZFNs are and how they work
They are artificial restriction enzymes generated by the fusion of zinc-finger DNA-binding domains to a DNA-cleavage domain i.e. the non-specific cleavage domain from the type IIS RE FokI from Flavobacterium okeanokoites (frog)
ZFNs recognise 4 codons (12 bases) and then cuts the 3’ end with the FOKI enzyme
What are some advantages and disadvantages of ZFNs?
- difficult to modify
- costly
- have to remake the whole system every time
+ very precise
What are TALENs and how do they work?
Xanthomonas bacteria (plant pathogen) express TAL arrays to bind and activate hot promoters
Xanthomonas uses 20-30 effector proteins that interfere with the plant immune system and various cellular processes
The TAL array is a series of DNA binding domains assembled to recognise specific DNA sequences and these can be constructed
Cut DNA with the FokI enzyme
What are some advantages and disadvantages of TALENs?
+ more precise than ZFN as each protein recognises 1 base rather than a codon
+ used in medical research
- difficult to patent
Why is CRISPR considered better than ZFNs and TALENS?
Because it is more equal than the other methods and can be used in a wide variety of organisms successfully
Give a brief history of CRISPR
Discovered in 1987
Was found to be an adaptive immune system in 2003
CRIPSR was targeted to DNA in 2006
Cas9 was found to create ds breaks in 2010
Genome editing using CRISPR in mammalian cells was completed in 2012
What does CRISPR stand for and when was it found?
Clustered regularly interspaced short palindromic repeats was found in E. coli by Yoshizumi Ito in 1987 using Sanger sequencing
Why did a yogurt company get involved in CRISPR research?
DANISCO found that phage infection in yogurt cultures was causing the bacteria to die
Archaea infected by phases in the past were found to keep a small part of the phage that infected them in a gene library and if infected by the same phage again, would check the CRISPR library and deploy Cas proteins to cut the phage before infection thus inactivating the viral DNA
Describe how Cas9 recognises and cleaves target DNA in viruses as part of the prokaryotic immune system
Protospacer adjacent motif (PAM, usually AGG spacer) prevents the Cas9 system from cutting the host DNA thus allowing recognition between self and non-self DNA
Describe what CRISPR is in prokaryotes and briefly describe how this works
An adaptive immune system means of protection from bacteriophages
Viral DNA is inserted into CRISPR arrays as spacers between the palindromic repeats
Transcription of these CRISPR arrays forms pre-crRNA
tracrRNA which is complementary to the palindromic repeats anneals to the pre-crRNA and RNase III cleaves between the palindromic repeats forming crRNA
A complex forms between crRNA, cas9 and tracrRNA and when another infection occurs the cas9 recognises a short sequence that is unique to the viral genome called PAM and will cut upstream of PAM thus neutralising the virus by preventing transcription
Allows for memory of a past infection
Describe how CRISPR/cas9 was adapted to work in eukaryotes
A linker can join the tracrRNA and crRNA together to form a single molecule in a hairpin loop called sgRNA which can form a complex with cas9 to cleave DNA (Charpentier, 2011)
sgRNA which is complementary to the target DNA is synthesised and inserted into the cell alongside cas9 which then forms a complex, the complex then reads the DNA, binding occurs and the DNA is cleaved
NHEJ (no template DNA needed/gives rise to indels thus changing the size of the DNA) or HDR (template DNA needed but doesn’t change the size of the DNA) then occurs
What is sgRNA?
Single guide RNA which is a construct/chimera of crRNA and tracrRNA
This simplifies the Cas9 system (Charpentier & Doudna, 2012)
What is crRNA?
CRISPR RNA
What is tracrRNA?
Trans-activating CRISPR RNA
What are the applications of CRISPR Cas9?
Targeted gene mutagenesis
Large scale chromosomal rearrangement
Genome scale gene knockout screening
Generation of transgenic organisms
Disease modelling
Gene therapy
Make any modification from 1bp to kilobases
Describe how base editing of AT to GC in genomic DNA was achieved without using DNA cleavage using CRRISPR
A hypothetical deoxyadenosine deaminase can change adenine to inosine which is read as guanine by polymerases
Changing just one base pair is more difficult than changing whole genes as it requires more precision
A deactivated Cas9 was bound to deoxyadenosine deaminase in a CRISPR complex Which opened the genomic DNA, delaminated the adenine in a ssDNA bubble, nicked the non-edited strand and DNA repair or replication replaces the inosine with guanine and the complementary base as cytosine
This can cause changes in amino acids and as such ~15,000 human diseases can theoretically be cured
Gaudelli et al 2018
What are the 3 applications of CRISPR beyond genome editing?
Gene regulation (switching genes on or off)
Cargo delivery (epigenetic manipulation such as demethylase delivery to remove methyl groups from DNA, or GFP delivery for visualisations
RNA cleavage
What is the experimental CRISPR workflow when designing a CRISPR experiment?
Design CRISPR guide RNAs
Order the synthetic guide RNAs and Cas9 and assemble into complex with modifications (small modifications to the 20bp sgRNA)
Delivery the CRISPR components through lipid transfection/electroporation (for advanced cells)/microinjection (for larger cells like eggs)/heat shock (E. coli and less advanced cells)/plant cell methods
Analyse the gene editing through cleavage assays/TIDE analysis/NGS/FACS
What is one of the problems with using CRISPR for gene therapy?
Can have off-target effects if used multiple times
Describe how the wild tomato was re-domesticated using gene editing
Before gene editing, selective breeding was used based on phenotypes such as sweetness/shelf-life/large fruit
CRISPR was used to insert genes into wild tomatoes to obtain these phenotypes much quicker than using selective breeding
Selective sweep (genetic hitchhiking) is where a new advantageous mutation becomes fixed in the population quicker and reduces variation in linked neutral sites as it becomes more frequent in a population
What are some examples of CRISPR edited foods?
Sicilian Rouge High GABA tomato provides more GABA amino acid which lowers blood pressure
Add vitamin D to tackle vitamin D deficiency in 1 Billion people
What is PERV and how has it been edited out of pigs using CRISPR Cas9?
Porcine endogenous retrovirus can infect humans and can affect the viability of pig organs for xenotransplantation
Pig organs are good for human transplants due to compatibility in size
Niu et al in 2017 engineered pigs in which all copies of PERVs were inactivated using CRISPR Cas9
What are the problems with trying to bring back extinct species and what are some examples?
Dwarf elephants are extinct and attempts to bring them back using gene editing may not work due to epigenetic changes to DNA and ancient DNA is too degraded to sequence
Explain how an altered gene can be spread using CRISPR and gene drives
A gene drive is a technique that propagates a specific allele through a population by altering the probability that it will be inherited
This would mean that both alleles are altered not just 1
Using HDR, a complex of Cas9, sgRNA and the altered allele can be inserted into the wild-type thus giving over a 50% chance of passing on the altered gene
Describe how Lyme disease can be combated using CRISPR
A gene drive that affects ticks can bear created and an altered gene can be inserted to make ticks not spread Lyme disease
A small trial was completed on an island
How can CRISPR and gene drives be used to save Kiwi birds?
There are many invasive species in New Zealand than kill kiwis and using a gene drive that was lethal would kill of the population in sites where they don’t belong thus saving the kiwis
What are the 2 types of gene drives?
Standard drives are the normal ones that have a fast spread, are indefinite and the allele frequency increases rapidly
Daisy drives are more complex, still spreads rapidly, however the allele frequency increases then decreases in a bell curve shape
Describe the use of CRISPR by companies
BASF licenced CRISPR-Cpf1 from MIT and Harvard
Monsanto licensed CRISPR to modify crops for herbicide resistance
MammothBiosciences uses CRISPR for disease detection
Many companies are trying to use CRISPR for gene therapy for conditions such as leukaemia which would involve taking blood cells, modifying them and returning them to the patients
Describe CRISPR babies
Twin baby girls were CRISPR edited to be HIV resistant by Dr He who altered their germ lines
This is very unethical and there were questions surrounding the informed consent procedure And there are many unknown future side effects
Rich people could create biologically elite babies which leads into Nazi ideologies and designer babies
Why could it be useful to reprogramme microbes using CRISPR?
E. Coli could be modified to make a synthetic microbe called E61 which has a synthetic genome like Synthia
3 codons have been removed in order to make it resistant to viruses
Amino acids that don’t naturally exist could be encoded into its genome which could lead to the production of new materials from drugs to biodegradeable plastics
Cambridge start up company called Constructive Bio
Describe some applications of CRISPR
Cancer immunotherapy by removing T cells from cancer patients, modifying them to be better at recognising and killing cancer cells and then returning them to the patient
AIDS treatment when anti-retroviral drugs won’t work due to the formation of the provirus inside T cells
Detection of viruses such as SARS-CoV-2
Genetic editing of plants to be disease resistant or have an increased product yield
