Gene editing

Question 1

give four examples of modern methods of gene editing

Answer 1

Meganuclease
ZFN (zinc finger nucleases)
TALEN (transcription activator like effector nucleases)
CRISPR/Cas9

Question 2

how do Meganucleases work?

Answer 2

their DNA recognition sequence is between 20-45 nucleotides
target sites tend to be introns and inteins
they intriduced dsDNA cutes which are repaired using HR and the intron/intein coding sequence as a template, thus introducing a newcopy of the intron/intein at the targeted site

Question 3

meganuclease mediated gene editing depends on what?
how is this a limiting factor?

Answer 3

Meganuclease-mediated gene editing depends on the presence of the corresponding DNA recognition site in the area of the genome where editing is needed, which is a serious limiting factor.
Currently several hundred meganucleases are known, and methods for evolving their specificity have been described, thus potential for developing a broad array of meganucleases exists. - however this still restricts us by the targets we can use

Question 4

biologically what is the function of meganucleases?

Answer 4

Biologically, phage meganucleases are thought to give competitive advantage to the phages that code them in mixed infections by cutting the genomic DNA of the coinfecting phages.

bacteriophage T4 codes for 15 different meganucleases, which take approximately 11% of the genome coding capacity.

Question 5

how do zinc finger nucleases work?

Answer 5

# Footnote

chimeric molecules composed of the nuclease domain of the FokI restriction enzyme and site-specific DNA-binding domain based on zinc-finger motifs - to tether the nuclease activity at a sequence of interest
These nucleases are modifiable so you can change the recognition sequence. These are specific. Recognise specific triplets in a specific sequence

ZFP belong to the class of DNA binding proteins. They bind to DNA and sometimes even to RNA and other proteins, through a finger-shaped fold, which is stabilized by zinc ions coordinated to a combination of cysteine and histidine residues.

Question 6

what is Fok1?

Answer 6

FokI is a dimeric-type IIS restriction enzyme isolated from Flavobacterium okeanokoites, which recognizes the 5′-GGATG-3′ sequence and introduces two single cuts 9 nt away from the 3′ end of its recognition sequence on the top strand and 13 nt away from the 5′ end of the bottom strand sequence (complementary to the listed one), thus collectively creating sticky ends with 4 nt-long overhang.

The mechanism of its action allows restriction to take place essentially in any sequence, as long as it is positioned at the precise distance from the recognition site bound by the DNA-binding domain

Question 7

how do TALENs work?

Answer 7

chimeric molecules comprised of the nuclease domain of the FokI restriction enzyme and site-specific DNA-binding domain based on the transcription activator–like effector proteins (in TALENs)
to tether the nuclease activity at a sequence of interest

bind 30-33 nucleotides, very targetable (can be engineered to recognise certain domains)
System is quite complicated, with a large protease that is difficult to deliver even by viruses.

Question 8

who won the Nobel Prize in Chemistry in 2020? (for CRISPR-Cas9)

Answer 8

awarded jointly to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing.”

for application of the CRISPR system to genetic modfication

Question 9

how was the CRISPR/Cas system first discovered?

Answer 9

In yoghurt factors, they had bacteriophage infections, luckily it was noticed some bacteria surivived, they were resistant to this type of bacteriophage. They have a prototype of the immune system - CRISPR/Cas

Question 10

how does CRISPR/Cas9 system work in bacteria and archaea?

Answer 10

New phage enters, injects its DNA. DNA is degraded upon entry and the loose info is bound by Cas1 and Cas2 and added to the CRISPR array. the CRISPR array has been expanded (new info, new spacer, ner repeat) and is transcribed. Cas proteins are formed and crispr RNA matures as Cas6/E cuts the loops of repeats (of pre crRNA). crRNA is loaded into the cascade proteins (Cas9)
when a known phage enters the cascade recognises the viral DNA (complementary sequence to the crRNA inside) and recruits the Cas 3 nucelease-helicase to cut the viral DNA

Question 11

what is different about the type 6 CRISPR/Cas system?
What can this be beneficial for?

Answer 11

it cuts RNA and not DNA

can be used for gene therapy when we dont want to destrythe genetic material of the cell or introduce it into the genome - just destroying the RNA of the faulty gene

Question 12

what are the differences between the wild type CRISPR/Cas9 compared to the one adapted for laboratory use?

Answer 12

The natural process involves both crRNA and tracrRNA
In the lab we use a fusion of the two, single guide RNA
- only the protospacer is changing according to our needs, this is a 20nucleotide sequence (statistically occuring once per genome, so off target effect rate is limited to how many homologs to this sequence)

Question 13

describe the mechanism of non homologous end joining

Answer 13

After double-strand break formation, the Ku70/80 heterodimer recognises the DSB, binds to the DNA ends and attracts DNA-PKCS.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a high affinity for Ku–DNA ends and, together with Ku, forms the DNA-PK complex

This activates the DNA-PK kinase activity, which leads to autophosphorylation
autophosphorylation activates artemis which then gains the ability to cut many DNA substrated at the boundaries between ss-dsDNA

DNA polymerase μ (Pol μ) and Pol λ interact with Ku through their N-terminal BRCA1 C terminus (BRCT) domains - these add nucleotides to the ends

DNA ligase IV and X-ray repair cross-complementing protein 4 (XRCC4) then ligates the ends together

Question 14

how does non homologous end joingint introduce mutations (insertions or deletions)?

Answer 14

during re ligation it can result in deletion or misalignment and fill in to enable complementary base binding

Question 15

describe the mechanism of homologous recombination

Question 16

what are the Four groups of site-specific nucleases can be employed to introduce sequence-specific dsDNA breaks?

Answer 16

Meganucleases
ZFN
TALEN
CRISPR/Cas9

Question 17

What CRISPR system is most useful at the moment?

Question 18

what are the potential gene editing application for CRISPR/Cas9?

Answer 18

gene surgery
drug development
animal models
genetic variation
materials
food
fuel

Question 19

give an example of an application of CRISPR/Cas for cancer

Answer 19

CRISPR/cas mediated knockout screening to identify the metastasis genes

Question 20

a chinese scientist was jailed for doing what in gene editing

Answer 20

A Chinese scientist claims to have helped make the world’s first genome-edited babies — twin girls, who were born this month. The announcement has provoked shock and outrage among scientists around the world.
He Jiankui, a genome-editing researcher at the Southern University of Science and Technology of China in Shenzhen, says that he impregnated a woman with embryos that had been edited to disable the genetic pathway HIV uses to infect cells.

Question 21

describe how CRISPR-Cas9 can be used in vivo as a gene editing treatment for transthyretin amyloidosis

Answer 21

• NTLA-2001 consists of a proprietary lipid nanoparticle (LNP) delivery system with liver tropism, carrying a single guide RNA (sgRNA) that targets human TTR and a human-codon–optimized mRNA sequence of Streptococcus pyogenes Cas9 protein
• Systemic administration of NTLA-2001 to six patients with hATTR amyloidosis with polyneuropathy was associated in each case with sustained reductions in the serum TTR protein concentration. NTLA-2001 treatment was associated with a dose-dependent effect. At day 28, the time at which the drug effect had reached its permanent nadir in preclinical studies, the mean reduction from baseline in serum TTR protein concentration was 52% in the group that received the lower dose (0.1 mg per kilogram) and was 87% in the group that received the higher dose (0.3 mg per kilogram). NTLA-2001 treatment was associated with adverse events of only mild severity.

modify gene expression of TTR in the liver using CRISPR-Cas9 system - measured protein expression and it was down 87%
associate only with mild adverse events
trial ongoing so yet to be approved

Question 22

what is NTLA-2001?

Answer 22

NTLA-2001 consists of a proprietary lipid nanoparticle (LNP) delivery system with liver tropism, carrying a single guide RNA (sgRNA) that targets human TTR and a human-codon–optimized mRNA sequence of Streptococcus pyogenes Cas9 protein

Question 23

what is ATTR amyloidosis?

Transthyretin amyloidosis

Answer 23

• Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart.
• After the onset of symptoms, ATTR amyloidosis is progressive, culminating in death within a median of 2 to 6 years after diagnosis in patients with amyloid cardiomyopathy and 4 to 17 years after symptom onset in patients with amyloid polyneuropathy in the absence of cardiomyopathy.

Question 24

what are the expected/potential clinical trials to ustilise CRISPR/Cas9 to treat disease?

Answer 24

• Cataract. Gene: Crygc
• Duchenne muscular distrophy. Gene: DMD
• β-thalassemia. Gene: HBB
• Cystic fibrosis. Gene: CFTR
• HIV. Region: HIV-1 LTR
• Epstein-Barr virus

Question 25

how can CRISPR/Cas9 be used to modify epigenetics?

Answer 25

Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state
You can do epigenetic modifcations resulting in transcriptional activation or repression by fusing Cas9 with histone demethylase (off) or acetyl transferase (on).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464239/

Question 26

how can you target two gene simultaneously with CRISPR/Cas9?

Answer 26

you can engineer inducible cas9 proteins from different species with different PAMs and different transcriptional modifiers like VP64 or COM and induce their expression by various signals eg bylight or tetracycline or certain other chemicals. One signal stops certain programs then another signal will do another or two signals to induce two programs simultaneously.

Lots of bacterial and eukaryoa have cas 9 and have protospacers different with length and sequence requirements enabling this

Question 27

how can we create a scenario where transcriptional activation/repression occurs by blue light?

Answer 27

During illumination CIB1 and CRY2 proteins bind together.
Fuse CIB1 protein with Cas9 (inactive form) and target it to certain genomic domain and then fuse transcriptional activator with CRY2. If you illuminate with blue light then you will have transcriptional activation
Simply by illuminating the cells you can activate it – if you stop illuminating the cell then transcription stops.

You can repress transcription in the same way - If you fuse it with a transcriptional repressor then blue light illumination will silence your gene.

Nihongaki et al., 2015
Arabidopsis cryptochrome 2 (CRY2)

Question 28

how can CRISPR/Cas be used to transcriptionally repress genes?

Answer 28

Create a Cas9 protein that has an inactive endonuclease domain that can still bind to the DNA but doesn’t elicit double stranded or even single stranded breaks
What is can do is present the binding of certain transcription factors or polymerase at the start of transcription or somewhere along the line, to block expression of certain genes without destroying it

catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas])

simply by using cas9 to block the promoter or polymerase

Making cas9 inducible by certain drugs then you have inducible silencing of genes – we can switch genes on and off without silencing them in vivo

Question 29

what is non homologous end joining?

Answer 29

# Footnote

Non-homologous end joining (NHEJ) is the primary pathway for the repair of DNA double-strand breaks (DSBs) throughout the cell cycle, including during S and G2 phases.
NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair(HDR), which requires a homologous sequence to guide repair.

NHEJ used to introduce mutations and deletions in CRISPR-Cas, you can predict what nucleotides will be inserted or deleted - a true knockout will have deletion by frame shift

Question 30

what three types of repair mechanisms are utilised by CRISPR-Cas9?

Answer 30

Homology-directed repair and non-homologous end joining are the two main types of DNA repair.

Microhomology-mediated end joining is a less common pathway that is sometimes deployed to repair DNA

Question 31

why is homology-directed repair (HDR) pathway favoured for treatment of monogenic diseases?

Answer 31

uses homologous donor DNA sequences from sister chromatids or foreign DNA to create accurate insertions, base substitutions between DSB sites or two DSBs, and other modifications.

Question 32

give 5 non homologous end joining subpathways

Answer 32

blunt ends
incompoatible 5’ ends
resection dependent compatible ends
incompatible 3’ ends
3’ phophorylated ends

Question 33

what nuclease is used in NHEJ?

Answer 33

When DNA resection is required for NHEJ, DNA-PKcs is recruited in complex with the endonuclease Artemis.
DNA-PKcs undergoes autophosphorylation and activates Artemis, which then gains the ability to cut many DNA substrates at the boundaries between single-strand and double-strand DNA (ss–dsDNA

Nuclease activity (also known as resection) is one method of ensuring that the two ends are compatible

Question 34

what polymerases are used in NHEJ?

Answer 34

NA polymerase μ (Pol μ) and Pol λ are the two members of the Pol X family polymerases that are involved in NHEJ in humans
These polymerases interact with Ku through their N-terminal BRCA1 C terminus (BRCT) domains

Question 35

what ligases are used in NHEJ

Answer 35

DNA ligase IV and X-ray repair cross-complementing protein 4 (XRCC4) are the most central components of NHEJ in eukaryotes

The N-terminal head domain of XLF interacts with the N-terminal head domain of XRCC4, and the XRCC4–XLF complex forms a sleeve-like structure around a DNA duplex

Question 36

what four ways can double stranded breaks be repaired?

Answer 36

non-homologous end joining (NHEJ) pathway, the alternative end joining (a-EJ) pathway, the single-strand annealing (SSA) pathway or by homologous recombination (HR).

Question 37

what is p53 binding protein 1?

Answer 37

p53-binding protein 1 (53BP1) is a chromatin remodeller and a positive regulator of NHEJ.

Question 38

what are the major differences between dsb repair pathways?

Answer 38

the requirement for substantial DNA end resection
* the complex of Artemis and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) can carry out some resection (typically <20 nucleotides), the NHEJ pathway does not require extensive end resection and the ends are mostly protected by the binding of Ku70–Ku80
* By contrast, carboxy-terminal binding protein interacting protein (CtIP) and the MRN (MRE11–RAD50–NBS1 (Nijmegen breakage syndrome protein 1)) complex are involved in extensive 5′ to 3′ resection of regions of the duplex to generate stretches of single-strand DNA (ssDNA) at DNA ends for a-EJ, SSA and HR

Question 39

what is the difference between the compoentns of the CRISPR-Cas9 system in wild type versus that used in the lab?

Answer 39

In the wild type there are two RNA components: tracer RNA and crRNA
In the lab we can use sgRNA which is fusion of the two RNAs, it is much easier to mainpulate

Question 40

what is the difference between introns and inteins?

Answer 40

Introns remove themselves at the RNA level; inteins remove themselves at the protein level.

Question 41

what are the benefits to gene editing by CRISPR rather than ZFN/TALENS?

Answer 41

Recognition of the DNA site in the CRISPR-Cas9 system is controlled by RNA–DNA interactions. This offers many advantages over ZFNs and TALENs, including easy design for any genomic targets, easy prediction regarding off-target sites, and the possibility of modifying several genomic sites simultaneously (multiplexing).

Question 42

what is the protospacer in the bacterial genome?

Answer 42

In a bacterial genome, CRISPR loci contain “spacers” (viral DNA inserted into a CRISPR locus) that in type II adaptive immune systems were created from invading viral or plasmid DNA (called “protospacers”).

Question 43

how can you edit several genes at the same time using CRISPR-Cas9?

Answer 43

inject several RNAs with Cas9 nuclease and it will be targeted to several genes cause ds breaks

Question 44

how can you used CRISPR-Cas9 mediated knockout screening to identify metastasis genes

Answer 44

Identify the genes that are responsible/important for metastasis or that can inhibit. Take non small small cell lung carcinoma and transfect it with Cas9-GFP by lentivirus so it is incorporated into the genome so the cell express cas9.
Then you inject it with a library of sgRNAs and transplant into a mouse. Then you look at the tumours and tumour metastasis
You know the site of insertion of the lentivirus so you can compare what genes are present in early and late tumuors – by sequencing those lentiviral insertion sites, looking at the genes that were switched off in metastasised cells.- idenitfying the genes important in metastasis

Question 45

how can transcription be repressed to silence genes by CRISPR-Cas9?

Answer 45

Create a Cas9 protein that has an inactive endonuclease domain that can still bind to the DNA but doesn’t elicit double stranded or even single stranded breaks
What is can do is present the binding of certain transcription factors or polymerase at the start of transcription or somewhere along the line, to block expression of certain genes without destroying it
If you make cas9 inducible by certain drugs then you have inducible silencing of genes – we can switch genes on and off without destroying them in vivo
Simply by blocking the promoter or dna polymerase.

Question 46

how have zinc finger nucleases been used for HIV resistance

Answer 46

The Zinc Fingers were engineered to disable the CCR5 gene in immune cells, allowing them to become resistant to HIV infection.