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BIOL 207 > Lec 6-3 > Flashcards

Lec 6-3 Flashcards

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
Q

CRISPR-Cas9 system gene therapy immunological challenges

A

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
Q

Immune privileged organs

Most progress for gene therapy has been made in the

A

Eyes

27
Q

Unknowns of CRISPR-Cas9 system for gene therapy

A

Extend of off target effects

Long term safety and the stability of gene editing

28
Q

CRISPR Forward genetics

A

Begins with a phenotype due to random mutations. The researcher uncovers what gene is responsible for the phenotype

29
Q

CRISPR reverse genetics

A

Begins with interest in a gene of unknown function. Then, induce mutations specifically in gene and check if phenotype occurs

30
Q

Forward genetics better for finding

A

Unknown genes

31
Q

Reverse genetics good if you

A

Have a strong hypothesis that a gene is important for a trait of interest

32
Q

Microsatellites

DNA Fingerprinting (DNA Profiling)

A

Short tandem repeats (STRs) variable number of copies of repeat sequences possessed by many organisms

33
Q

Microsatellites are detected by

A

PCR

34
Q

Fragments represented as peak on a graph are either

A

Homozygotes- single tall peak

Heterozygotes- Two shorter peaks

35
Q

DNA fingerprinting can be used to identify

A

People

36
Q

DNA fingerprinting methods

A

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
Q

DNA fingerprinting results

A

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
Q

A DNA profile represents

A

The pattern of DNA fragments produced by performing PCR on the STR loci

39
Q

Number below each peak on a DNA profile is

A

Number of STRs and DNA fragment

40
Q

DNA fingerprinting can be used to determine the presence of

A

A suspect at a crime scene

41
Q

Fingerprinting was used to identify

A

People who died during 9/11

42
Q

DNA barcoding

A

Technique to identify different species using DNA

Used genes that have high interspecies variability (between species)
and low intraspecies variability (within species)

43
Q

DNA barcoding can be used to identify

A

The species of meats sold at markets or restaurants

Are food labels accurate?

44
Q

DNA barcoding applications

A

The species of meats sold at markets

Species of plants or insects

Diet of organism

Human matrilineal lineages

Animal remains

45
Q

DNA barcoding workflow

A

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

BIOL 207 (31 decks)

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