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Regulation of Gene Expression > Ferrari: Lecture X > Flashcards

Ferrari: Lecture X Flashcards

Genome Editing

1
Q

What is the most current way that we have in clinic to correct diseases with a known mutation from a genetic point of view?

A

if we are adding, vectors are used by adding gene in stem cells in vivo

if we are editing the genome, genome editing is used and made possible because of viral vectors that switches the correct copy of the gene for the mutated one

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2
Q

What is genomic substitution called?

A

homologous recombination, and it occurs at a low frequency

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3
Q

What are the most used molecular tools in research and clincal field?

A

Zinc Finger Nucleases (ZFN)
Meganucleases (different versions derived from ZFN

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4
Q

What is a nuclease?

A

molecule that can cut DNA

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5
Q

Transcription Activator Like Effector Nuclease (TALEN)

A

nuclease that can cut DNA and the genome

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6
Q

Clustered Regularly Interspaced Palindromic Repeat (CRISPR/Cas9)

A

most famous system

*we MUST remember the acronym

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7
Q

How do we know which molecular tool to use?

A

we have a summary guide where we can choose the tool based on different considerations

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8
Q

Why is CRISPR/Cas9 used in many labs?

A

it is cheap, works well, & very efficient

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9
Q

What makes ZFN, meganucleases, and TALEN not as useful as CRISPR/Cas9?

A

more expensive and more complicated

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10
Q

What is important to remember in regards to molecular tools that are very efficient?

A

there is a risk that off-target sites are modified along with the target site

methods to detect the off-sites would need to be synthesized

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11
Q

Describe the genome editing approach:

A

dsDNA is cut
nucleases induce double strand breaks (DSB)

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12
Q

What systems repair DSB?

A

nonhomologous end joining (NHEJ)
homology direct repair (HDR)

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13
Q

NHEJ

A

cut ends are repaired by cellular machinery

bases can be added or cut (called idels)

the rejoining of ends is not precise

gene function is disrupted

if 1 or 2 codons are deleted but the sequence is still in frame, we might have the right product (if indels does not occur in the region) otherwise a gene is knocked-out

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14
Q

What is the result of NHEJ?

A

gene disruption

potentially large insertions

if nuclease is cut twice, inversions are possible (instead of losing DNA, the pieces are flipped)

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15
Q

HDR

A

donor template must be provided

piece of DNA is added very precisely (where the genome is cut)

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16
Q

What is targeted gene addition in HDR?

A

if a gene is mutated (purple arrow), and the correct copy of the gene is provided, the gene is corrected because of substitution

the exact location where we are putting the gene is known

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17
Q

What is an advantage of doing gene addition in the manner where we put a piece of DNA and express it in the genome?

A

if gene addition of just the coding region targeting the promoter of the gene is naturally expressed in the gene, the expression occurs in a more physiological way from the endogenous promoter

insertion or mutagenesis is also avoided since precise location is known

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18
Q

What is the preferential pathway of a DSB?

A

NHEJ, even if a donor template is provided

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19
Q

What triggers HDR?

A

nucleases, so it does not occur naturally

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20
Q

List the molecular tools available for genome editing:

A

zinc finger nucleases
TALEN
CRISPR/Cas9

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21
Q

zinc finger motif

A

present naturally in proteins & it is a DNA binding domain

some TFs contain the ZF domain

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22
Q

zinc finger nuclease

A

artificial protein, not present in nature

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23
Q

How do you form ZFN I?

A

combining DNA binding domain of ZF to a nuclease

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24
Q

What provides the nuclease domain in the ZFN?

A

restriction enzyme, FokI

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25
Q

What can FokI do?

A

cut DNA on both strands, but it is sequence independent

26
Q

Where does the cutting of the restriction enzyme occur?

A

far away from where the enzyme binds the DNA

27
Q

The zinc finger nucleases are ____

A

modular recognition proteins

FokI is fixed, but the nuclease can be designed to vary the design of the ZF domain

28
Q

ZFNs are ___________ in inducing genome sequence changes in all the mammalian cells in rodents, human, and most any types of cells.

A

very efficient

29
Q

What are some cons to ZFN?

A

designing the protein is hard and this can cause issues in validating if the ZFN is effective in cutting the target

30
Q

Review the Zinc Finger characteristics:

A
31
Q

Analyze and Describe the ZFN:

A

ZFN work in couple

they bind the DNA target as a dimer (right ZFN and left ZFN)

FokI domain is the common domain for both ZFN and it is also the cleavage domain

2 DNA binding sites

spacers are composed of 5-7 bp for the FokI cleavage site

usually you start from the DNA target and on the basis of the sequence you design the ZF

32
Q

TALEN are _____

A

artificial proteins not present in nature, but they are derived from a natural molecule which is the transcriptional activator-like effectors (TALE)

33
Q

Where are TALE proteins present?

A

bacteria which infect plants, in particular Xanthomonas

34
Q

What does injection of TALE in plants do?

A

activate the expression of gene that helps bacteria for replication and spread

they activate transcription, which helps bacteria activate genes from the host for replication and life cycle

35
Q

How do we make TALEN?

A

TALE heart (N-terminal translocation domain)

C-terminal activator domain for activation of plant genes

central repeat for binding domain (composed by 15-19 repeats with some conserved residues)

36
Q

How do we modify the original protein to make our gene editing tool TALEN?

A

TALEN works as a dimer (2 TALEN)

FokI domain

DNA binding domain

N-terminal domain

37
Q

Analyze and Describe TALEN:

A

2 TALE (right TALE & left TALE)

FokI cleavage domain

spacer

TALEN offer the possibility to have a longer recognition sequence for DNA compared to ZFNs (allows for precise targeting of a gene)

TALEN are difficult to synthesize (more specific compared to ZFNs)

38
Q

What are some genetic diseases where we can use ZFNs and the HDR pathway?

A

X-linked SCID (immunodeficiency)
Haemophilia B
Sickle Cell Disease

1st and 3rd affect the hematopoietic system

39
Q

How can we work to only disrupt the gene, not correct?

A

ZFN (KO via NHEJ)

tested in a clinical trial of HIV resistance in CCR5 KO in T cells

40
Q

What is a good way we could prevent infections of T cells in humans?

A

using NHEJ of ZFNs (there are current clinical trials ongoing)

clinical trial: KO endogenous TCR in generation of tumour specific T-cell like the CAR-T cells in oncology

41
Q

Describe CRISPR/Cas9 origin and history:

A

1987: unknown biological significance of sequences in CRISPR locus E. coli

2005: 1st demonstration of potential connection between CRISPR’s locus and an antiviral immune defense

2007: CRISPRs found in 40% to 90% of all bacterial and archaeal species

2007: Streptococcus thermophilus CRISPR spacers confer potent resistance to bacterial viruses (bacteriophage) bearing matching DNA sequences and bacteria could actively vaccinate themselves

2008: central role of noncoding CRISPR RNA (crRNA) was discovered

42
Q

What do we know about the bacterial vaccination system?

A

different from CRISPR/Cas9 system: almost all archaebacteria use small ncRNAs to protect themselves from viruses

it is similar to adaptive immunity in mammals

bacteria cells have a memory of infections because they carry a record of past exposures that is used to protect against future exposures

43
Q

Describe how a bacteriophage enters the bacterium:

A

short viral DNA sequence is integrated (different colors are previous infections from different phages; the orange alters the specific phage’s sequences and are not from old infection)

CRISPR locus is transcribed

pre-crRNA is generated, transcribed, processed, and bound to Cas protein

at the next viral infection site, cell will inject inside the genome and the ds viral DNA will be cleaved because of the small crRNA that are homologous to the phage due to previous infection

44
Q

Why can the bacteriophage enter the bacterium?

A

because the CRISPR/Cas9 system is based on antisense RNAs memory due to the memory signatures based on past invasions

45
Q

Describe CRIPSR/Cas9 RNA-endonuclease complex:

A

based on the adaptive immune system of Streptococcus Pyogenes SF370

system can target and cleave target DNA through a programmable short guide RNA binding to its complementary DNA strand and a Cas9 system

46
Q

Describe the features of the CRISPR/Cas9 system:

A

Cas gene is produced by the same locus

there is a leader sequence (in L)

repeats are in different colors

spacers are black

47
Q

How does the CRISPR/Cas9 system work molecularly?

A

CRISPR locus is transcribed and generates CRISPR RNA precursor (pre-crRNA)

seperate ncRNA and tracrRNA are transcribed and anneals to pre-crRNA repeats

strands of the duplex are cleaved y host RNAse III to generate different crRNA

crRNA 5’ is trimmed

ds complex is made by crRNS and the tracrRNA

crRNA engages an effector complex containing the Cas protein, which is transcribed from the CRISPR locus

Cas9: crRNA-tracrRNA complex can recognize and cleave target DNA sites

48
Q

What does the Cas9 protein recognize?

A

protospacer adjacent motif (PAM) of target DNA, that is usually adjacent to the RNA-binding site and made by a few nucleotides

49
Q

To make an engineered CRISPR/Cas9 system, we only keep 2 portions: _____ & _____.

A

crRNA and tracrRNA

50
Q

What is is called when crRNA and tracrRNA are fused?

A

sgRNA, which is a single guide RNA

51
Q

What can sgRNA do?

A

form a complex with Cas9 and target the DNA site to cleavage

52
Q

What happens once there is a match between sgRNA, and DNA?

A

Cas9 cleaves DNA where the PAM sequence is located

53
Q

Watch the following video of CRISPR/Cas9:

A

https://www.youtube.com/watch?v=2pp17E4E-O8

54
Q

When a guide is designed, we have specificity of recognition, but it does not guarantee the cleavage of the DNA target, so it must also be present in the ___ sequence in the target DNA in order to be recognized by the ___ protein.

A

PAM

Cas

55
Q

What kind of genomic sequences does Cas9 target?

A

sequences containing the tri-nucleotide (NGG, which N can correspond to A, T, C, or G) protospacer adjacent motif (PAM) sequence close to the gRNA

*we can change the Cas system because NGG is the PAM specific for the system of streptococcus pyogenes

56
Q

What is the role of NGG PAM that is known by following it to the binding site?

A

Cas9 cleaves DNA 3 or 4 bp upstream of the NGG PAM and the double strands generated are repaired by NHEJ or HDR

57
Q

Is it easy to reprogram CRISPR/Cas9?

A

yes, it requires only the presence of PAM in the target DNA and the change of the 1st 20 bases in the gRNA

when a donor template is given , the system can achieve almost precise genome correction

58
Q

Who developed the CRISPR/Cas system for genome editing?

A

Emmanuelle Charpentier and Jennifer A. Dounda

59
Q

What are some CRISPR/Cas9 application?

A

genome editing

genome-wide screening

gene regulation

human therapeutics

ecological engineering

RNA manipulation

60
Q

What are some examples of some cell types and organisms that have been engineered using Cas9?

A

HEK293
crop plants
organoids
hESCs
iPSCs

Regulation of Gene Expression (28 decks)

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