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Alex's Genetics > Lecture 5a > Flashcards

Lecture 5a Flashcards

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1
Q

When we are using Crispr-Cas9 as a tool, what are we using it for?

A

We are making modifications in the genome.

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2
Q

When we are using Crispr-Cas9 as a tool, what serves as the “Guide RNA”?

A

The crRNA and the tracrRNA make up one guide RNA.

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3
Q

If we are modifying the genome, what is the role of the Cas9 protein?

A

It cleaves both strands next to the genomic target sequence.

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4
Q

How can we obtain the guide RNA we need to make modifications to a genome?

A

Companies will synthesize the guide RNA, which can then help the crispr-cas complex to find the specific gene we want to alter/delete.

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5
Q

Where does the Cas9 protein cleave?

A

It cleaves the chromosome in front of and behind the gene.

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6
Q

What happens to the gene after it is cleaved by Cas9?

A

The gene will float away and tend to get lost.

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7
Q

What happens to the remainder of the broken chromosome after Cas9 has cleaved the gene out?

A

The cell uses Non-Homologous End Joining (NHEJ) to rejoin the chromosome.

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8
Q

How do we determine if we successfully deleted a gene?

A

Polymerase Chain Reaction (PCR).

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9
Q

What can happen in PCR if the gene is big and the primers are far apart?

A

The PCR would not work.

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10
Q

How does PCR allow us to determine if we successfully deleted a gene?

A

We can amplify a section of the homologous chromosome where the deleted gene would have been. The products should contain none of the gene we cleaved out.

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11
Q

What are the products of PCR?

A

DNA nucleotide sequences of a section of a chromosome.

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12
Q

T/F: Some forms of Homology-Directed Repair require only a single strand of DNA as a template for the repair of a double strand break.

A

True!

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13
Q

Generally, how does single-stranded HDR work?

A

1) There will be a single strand of DNA, which will contain a specific sequence we want to add. On the sides of this specific sequence, there will be sequences identical to the chromosome we are adding the sequence into.
2) A double-stranded break will be made using CRISPR-Cas9.
3) Then, HDR will insert the foreign DNA sequence into the double-stranded DNA break.

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14
Q

What is the product of single-stranded HDR?

A

A foreign DNA sequence will be inserted into the chromosome where the double-stranded break occurred.

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15
Q

Why do we see general diagrams for HDR?

A

The specific steps of HDR have no clearly been worked out.

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16
Q

Explain the process for using CRISPR-Cas to insert a gene into a chromosome.

A

1) First, we need to obtain the gene of interest. If a gene is less than 10-kb (small), we can amplify the gene through PCR to obtain it.
2) Cas9 protein makes a double-stranded break in the DNA.
3) The gene is placed in the middle of the break.
4) HDR uses ultramers to ligate the DNA.

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17
Q

What are ultramers?

A

They are pieces of single-stranded DNA 180 nucleotides long. 90 of the nucleotides match the sequence of the gene we are inserting. 90 of the nucleotides match the sequence of the chromosome we are inserting the gene into.

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18
Q

How many ultramers are needed to insert a single gene?

A

2

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19
Q

What’s another name for ultramers?

A

Long oligonucleotides

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20
Q

What do we always need to do after we insert a gene into a chromosome?

A

We need to use PCR to amplify the insertions junctions to confirm that the gene has been inserted.

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21
Q

Describe the process for using CRISPR-Cas to replace a gene.

A

1) Cas9 protein cleaves the original gene on both sides of it.
2) The new gene is placed in between the double-stranded break.
3) Homology-Directed Repair uses ultramers on both sides of the new gene to ligate the DNA.

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22
Q

What do we call it when we use CRISPR-Cas to replace a gene?

A

A knock-in.

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23
Q

What are the insertions junctions on a chromosome?

A

They are where the ends of a newly inserted gene connect with the original chromosome.

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24
Q

What are transgenes?

A

When we insert a gene that previously didn’t exist in a species.

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25
Q

If we want to insert a gene in a mammal, where should we do it? Why?

A

In the ROSA26 locus. This locus is in the gene for a long non-coding RNA, thus it is not very important.

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26
Q

What has been found to be a great place in mammals to express transgenes?

A

The ROSA26 locus

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27
Q

What are two ways to cleave at ROSA26?

A

We can use Cas9 to cleave on both ends of the ROSA26 locus or we can cleave it right down the middle.

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28
Q

How do we check for success of HDR?

A

PCR amplification at the insertion junctions to confirm that the gene has been inserted.

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29
Q

T/F: Many genes are too large to PCR amplify.

A

True!

30
Q

What is difficult about PCR with eukaryotic genes?

A

Most eukaryotic genes are unexpectedly large, especially in higher eukaryotes, thus we cannot use PCR to amplify most of these genes.

31
Q

Describe the hybridization of mRNA to DNA for most bacterial genes.

A

1) Heat the DNA to denature the strands.
2) Then, we add mRNA and let it cool slowly. Because there are several copies of the mRNA, it will outcompete the DNA.
3) The mRNA binds to the DNA, forming an R loop.

32
Q

When we denature DNA strands, what are we doing that separates the strands?

A

We are breaking the hydrogen bonds.

33
Q

How are we able to visualize the mRNA bound to the DNA?

A

We can visualize it using an electron microscope.

34
Q

What does an electron microscope allow us to do with genes?

A

We are able to map genes using electron microscopes.

35
Q

Describe the Hybridization of other mRNA to DNA for most eukaryotic genes.

A

1) There are discontinuous regions of DNA that are complementary to mRNA. In between these regions is an intron.
2) The DNA is heated to separate the strands.
3) mRNA is added and binds to the DNA. An ugly ass loop is formed with 2 R loops, intron DNA, and mRNA.

36
Q

What was the importance of looking at hybridization of other mRNA to DNA for most eukaryotic genes?

A

We were able to see that a section of the mRNA had been deleted, which allowed for the discovery of the intron.

37
Q

Who discovered introns?

A

Roberts and Sharp. They received a Nobel Prize for this.

37
Q

What was creating the ugly ass loop in the hybridization with eukaryotic genes?

A

There was an intron in the pre-mRNA that had been previously spliced out.

38
Q

After splicing, what can we say about genes regarding size?

A

Genes may be huge initially, but the mRNA is much smaller after splicing.

39
Q

What is the mRNA that is directly made from a gene?

A

Pre-mRNA.

40
Q

What happens to the pre-mRNA that it becomes mRNA?

A

It undergoes splicing, in which the introns get spliced out.

41
Q

What are exons?

A

The parts of the mRNA that do NOT get spliced out.

42
Q

What are introns?

A

The parts of the mRNA that are spliced out.

43
Q

What codes for the protein?

A

mRNA.

44
Q

What 3 things is the mRNA made of?

A

A cap, exons, and a poly-A tail.

45
Q

What is RNA splicing?

A

The process of removing introns.

46
Q

How many mechanisms are there for RNA splicing?

A

3 mechanisms

47
Q

What are the names of the mechanisms for RNA splicing?

A

Group I, Group II, and Spliceosome Splicing.

48
Q

What is Spliceosome splicing? What is its requirement?

A

The most common mechanism for RNA splicing. It is the only mechanism that requires splicing proteins.

49
Q

What are Group I and Group II able to do? How common are they?

A

They are able to ‘self-splice’. They are rare.

50
Q

Which RNA splicing mechanisms are basically the same except for the requirement of splicing proteins?

A

Group II and Spliceosome Splicing are basically the same mechanism.

51
Q

What does Group I splicing create?

A

It creates a linear intron that is spliced out.

52
Q

What do Group II and Spliceosome Splicing create?

A

They create a lariat intron that is spliced out.

53
Q

How do we define the intron RNA with spliceosome splicing?

A

The intron RNA is defined by particular sequences within the intron and at the intro-exon boundaries.

54
Q

What are the 3 main sites we see for spliceosome splicing?

A

The 5’ splice site, the branch site, and the 3’ splice site.

55
Q

What sites serve as the recognition sites for the binding of the spliceosome?

A

The 5’ splice site and the branch site.

56
Q

What do we mean when we say there is sequence conservation in spliceosome splicing?

A

There are only 3 markings throughout the whole intron for splicing. The sequences are highly conserved.

57
Q

What is the function of the 5’ splice site and the branch site?

A

They serve as recognition sites for the binding of the spliceosome.

58
Q

What is at the branch site that is important?

A

The adenine

59
Q

What is the function of the adenine at the branch site?

A

It is the site where loops occur to form a lariat.

60
Q

What is the only site that is not next to an exon?

A

The branch site.

61
Q

What is capping?

A

A GMP (RNA nucleotide) is attached to the 5’ end of RNA with phosphate-to-phosphate covalent bond.

62
Q

What is the importance of capping and tailing?

A

It makes the RNA more stable and is essential for translation.

63
Q

Why is capping necessary for translation?

A

The cap is the spot that the ribosomes recognize.

64
Q

What is Polyadenylation?

A

The addition of a tail consisting of multiple Adenines. This is a PolyA tail.

65
Q

What is the Polyadenylation signal in higher eukaryotes?

A

AAUAAA

66
Q

What recognizes the polyadenylation signal?

A

Polymerase

67
Q

Describe the process of tailing.

A

1) Polymerase recognizes the polyadenylation signal.
2) Endonuclease cleavage occurs about 20 nucleotides downstream (before) from the sequence.
3) PolyA-polymerase adds Adenine nucleotides to the 3’ end.

68
Q

What are some features about the PolyA tail length?

A

The length of it varies between species.

The length can vary from a few dozen adenines to several hundred.

69
Q

What type of RNAs do capping and tailing occur in?

A

Capping and tailing occur in eukaryotic RNAs.

Alex's Genetics (38 decks)

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