Gene Editing

Question 1

What are 3 relatively new methods of gene editing?

Answer 1

ZFNs

TALENs

CRISPR/Cas9

ZFNs and TALENs came before CRISPR

Question 2

What do those 3 gene editing tools have in common?

Answer 2

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

Question 3

Describe what ZFNs are and how they work

Answer 3

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

Question 4

What are some advantages and disadvantages of ZFNs?

Answer 4

• difficult to modify
• costly
• have to remake the whole system every time

+ very precise

Question 5

What are TALENs and how do they work?

Answer 5

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

Question 6

What are some advantages and disadvantages of TALENs?

Answer 6

+ more precise than ZFN as each protein recognises 1 base rather than a codon

+ used in medical research

• difficult to patent

Question 7

Why is CRISPR considered better than ZFNs and TALENS?

Answer 7

Because it is more equal than the other methods and can be used in a wide variety of organisms successfully

Question 8

Give a brief history of CRISPR

Answer 8

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

Question 9

What does CRISPR stand for and when was it found?

Answer 9

Clustered regularly interspaced short palindromic repeats was found in E. coli by Yoshizumi Ito in 1987 using Sanger sequencing

Question 10

Why did a yogurt company get involved in CRISPR research?

Answer 10

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

Question 11

Describe how Cas9 recognises and cleaves target DNA in viruses as part of the prokaryotic immune system

Answer 11

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

Question 12

Describe what CRISPR is in prokaryotes and briefly describe how this works

Answer 12

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

Question 13

Describe how CRISPR/cas9 was adapted to work in eukaryotes

Answer 13

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

Question 14

What is sgRNA?

Answer 14

Single guide RNA which is a construct/chimera of crRNA and tracrRNA

This simplifies the Cas9 system (Charpentier & Doudna, 2012)

Question 15

What is crRNA?

Answer 15

CRISPR RNA

Question 16

What is tracrRNA?

Answer 16

Trans-activating CRISPR RNA

Question 17

What are the applications of CRISPR Cas9?

Answer 17

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

Question 18

Describe how base editing of AT to GC in genomic DNA was achieved without using DNA cleavage using CRRISPR

Answer 18

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

Question 19

What are the 3 applications of CRISPR beyond genome editing?

Answer 19

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

Question 20

What is the experimental CRISPR workflow when designing a CRISPR experiment?

Answer 20

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

Question 21

What is one of the problems with using CRISPR for gene therapy?

Answer 21

Can have off-target effects if used multiple times

Question 22

Describe how the wild tomato was re-domesticated using gene editing

Answer 22

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

Question 23

What are some examples of CRISPR edited foods?

Answer 23

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

Question 24

What is PERV and how has it been edited out of pigs using CRISPR Cas9?

Answer 24

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

Question 25

What are the problems with trying to bring back extinct species and what are some examples?

Answer 25

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

Question 26

Explain how an altered gene can be spread using CRISPR and gene drives

Answer 26

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

Question 27

Describe how Lyme disease can be combated using CRISPR

Answer 27

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

Question 28

How can CRISPR and gene drives be used to save Kiwi birds?

Answer 28

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

Question 29

What are the 2 types of gene drives?

Answer 29

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

Question 30

Describe the use of CRISPR by companies

Answer 30

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

Question 31

Describe CRISPR babies

Answer 31

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

Question 32

Why could it be useful to reprogramme microbes using CRISPR?

Answer 32

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

Question 33

Describe some applications of CRISPR

Answer 33

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