Lec 6-3 Flashcards

(45 cards)

1
Q

CRISPR/Cas genome editing capabilities

A

Edit any genome

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2
Q

CRISPR-Cas background

A

Started with the question of what are the ‘weird’ sequences in genomes of bacteria and archaea

Contains spacers and palindromes

Spacers had DNA sequences of foreign origin

What were previously thought of as DNA repair proteins were discovered to be strictly associated with CRISPR

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3
Q

CRISPR stands for

A

Clustered regularly interspaced short plaindromic repeats

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4
Q

CRISPR Cas system in bacteria

A

Evolved in bacteria to combat invading DNA elements, similar to an immune system

Functions as a type of organismal memory of past invaders

Leads to the targeted destruction of invading DNA

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5
Q

How do bacteria remember past invaders

A

They create CRISPR arrays in the bacterial genome (Acquisition phase)

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6
Q

CRISPR arrays have two parts

A

Protospacers- Unique spacers (sequence derived from past invaders)

Palindromic repeats flank protospacers (Read the same forward or reverse)

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7
Q

How do bacteria use their memory to target/destroy future invaders

A

They express complementary RNA to similar invaders (Expression phase)

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8
Q

Expression phase

A

CRISPR array is transcribed into a single long precursor RNA called the Pre-cRNA

Pre-crRNA are cleaved by Cas nuclease proteins into short crRNA with unique protospacer sequence

crRNQ +tracrRNA + Cas( form complex

Complex binds to complementary DNA of new invaders

Complex cleaves causing double stranded breaks

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9
Q

Molecular aspects of targeting/cleavage in CRISPR

A

crRNA contain 20-50nt of invader homology

Protospacer adjacent motif is needed downstream of the spacer

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10
Q

CRISPR-Cas can targer

A

very unique DNA sites

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11
Q

PAM sequence in CRISPR-Cas9 system is

A

5’-NGG-3’

Short 3nt sequence

Occurs very frequently throughout a genome

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12
Q

CRISPR-Cas9 is most widely used in

A

Molecular genetics

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13
Q

Natural system requires

A

crRNA and tracrRNA to pair and bind with Cas9 to make effector complex

Researchers combine crRNA, tracrRNA into one single guide RNA (sgRNA)

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14
Q

sgRNA contains

A

20nt seed sequence that can direct the effector complex to any specific DNA sequence

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15
Q

Purpose of PAM

A

Enables Cas9-mediated recognition and cleavage of target DNA

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16
Q

What happens to cleaved DNA

A

DNA repair

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17
Q

Two DNA repair mechanisms exist to fix double stranded breaks

A

Nonhomologous end joining (NHEJ)

Homology directed repair (HDR)

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18
Q

Nonhomologous end joining

A

If homologous template is not present

Used when cells are in G1 and no sister chromatid available as a template

Error prone and often leads to deletions, insertions, and translocations

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19
Q

Homology directed repair

A

If a homologous template is present

Normally it is the sister chromatid after DNA replication

20
Q

Homology directed repair for transgenics (organism that is altered by the introduction of one or more foreign DNA sequence)

A

Researchers provide the homologous template called donor DNA

Insert desired new sequences into a gene

21
Q

Disadvantage of HDR

A

Not very efficient
Low success rate

22
Q

Ethical challenges of CRISPR-Cas9 for gene therapy

A

Somatic gene therapy is ethically more permissible

Germ-line gene editing is not currently possible due to ethical reasons

23
Q

Delivery of materials into target cells challenges for CRISPR-Cas9 for gene therapy methods

A

Viral vector delivery

Non-viral delivery

24
Q

Delivery of materials into target cells challenges for CRISPR-Cas9 for gene therapy

Will these delivery methods work

A

Many cells must be edited because gene editing with homology directed repair is very inefficient

25
CRISPR-Cas9 system gene therapy immunological challenges
Immune response against Cas9 protein can destroy protein function because Cas9 is bacterial Immune response to delivery mechanisms Can lead to autoimmune-like disease/symptoms
26
Immune privileged organs Most progress for gene therapy has been made in the
Eyes
27
Unknowns of CRISPR-Cas9 system for gene therapy
Extend of off target effects Long term safety and the stability of gene editing
28
CRISPR Forward genetics
Begins with a phenotype due to random mutations. The researcher uncovers what gene is responsible for the phenotype
29
CRISPR reverse genetics
Begins with interest in a gene of unknown function. Then, induce mutations specifically in gene and check if phenotype occurs
30
Forward genetics better for finding
Unknown genes
31
Reverse genetics good if you
Have a strong hypothesis that a gene is important for a trait of interest
32
Microsatellites DNA Fingerprinting (DNA Profiling)
Short tandem repeats (STRs) variable number of copies of repeat sequences possessed by many organisms
33
Microsatellites are detected by
PCR
34
Fragments represented as peak on a graph are either
Homozygotes- single tall peak Heterozygotes- Two shorter peaks
35
DNA fingerprinting can be used to identify
People
36
DNA fingerprinting methods
DNA samples are collected (One of which a microsatellite) and subjected to PCR The length of the DNA fragments produced by PCR depends on the number of copies in the microsatellite sequence
37
DNA fingerprinting results
The fragments are separated by gel electrophoresis Different sized fragments appear as different bands If the bands align they belong to the same person
38
A DNA profile represents
The pattern of DNA fragments produced by performing PCR on the STR loci
39
Number below each peak on a DNA profile is
Number of STRs and DNA fragment
40
DNA fingerprinting can be used to determine the presence of
A suspect at a crime scene
41
Fingerprinting was used to identify
People who died during 9/11
42
DNA barcoding
Technique to identify different species using DNA Used genes that have high interspecies variability (between species) and low intraspecies variability (within species)
43
DNA barcoding can be used to identify
The species of meats sold at markets or restaurants Are food labels accurate?
44
DNA barcoding applications
The species of meats sold at markets Species of plants or insects Diet of organism Human matrilineal lineages Animal remains
45
DNA barcoding workflow
Extract mystery DNA Amplify COI region with PCR Check on gel and purify column DNA (sanger) Sequence product Obtain a DNA sequence Identify your likely species using DNA sequence database