lecture 31

Question 1

surface barriers, cellular barriers, and inflammation are examples of what?

Answer 1

innate immunity

Question 2

lymphocytes, T-cells, and B-cells are examples of what?

Answer 2

acquired immunity

Question 3

when is acquired immunity “acquired?”

Answer 3

when a cell reacts to a foreign object/infection and keeps memory of it

Question 4

how does viral DNA begin to be made in an bacterial cell?

Answer 4

a bacteriophage made of protein-coated DNA anchors itself in the cell wall of a bacteria cell and injects its DNA into the cell through a shealth; the organelles begin making this viral DNA

Question 5

how does a bacteriophage-infected cell spread the virus to other cells?

Answer 5

the cell becomes so full of bacteriophages that its organelles have created that it bursts open, releasing the new bacteriophages to infect other cells

Question 6

what does naturally occurring CRISPR in bacterial cells do?

Answer 6

acts as the bacteria’s immune system that protects them from viruses, especially bacteriophages, by storing a memory of previous viral infections and using that memory to recognize and fight future infections

Question 7

what does CRISPR stand for?

Answer 7

clustered regularly interspaced short palindromic repeats

Question 8

what is meant when it is said that palindromic repeats read the same way backwards and forwards?

Answer 8

comparing it base-by-base, it reads the same in either direction. GAATTC is an example of a palindromic repeat because the complimentary base pairs when read in the same 5’ to 3’ direction are GAATTC even though it is CTTAAG that binds

Question 9

what shape do palindromic repeats form?

Answer 9

hairpin structure

Question 10

what is spacer DNA complementary to?

Answer 10

viral DNA

Question 11

what are the types of proteins made from CAs genes?

Answer 11

endonucleases and helicases

Question 12

what is the order of CRISPR RNA?

Answer 12

CAs genes, repeats, unique spacer, repeats, unique spacer, etc.

Question 13

what is spacer DNA?

Answer 13

unique DNA segments between each set of palindromic repeats that are derived from viral DNA that has been previously encountered and entered into the genome. it holds the “memory” of viruses.

Question 14

where are CAs genes located relative to the repeats and spacers?

Answer 14

CAs genes are upstream

Question 15

what do CAs genes do?

Answer 15

produce proteins(endonucleases and helicases) that carry out the immune functions of CRISPR

Question 16

what type of molecule is Cas9?

Answer 16

protein –> enzyme –> endonuclease

Question 17

in what structure is lab-created CRISPR/cas-9?

Answer 17

plasmid

Question 18

in a bacterial cell, once the viral DNA is injected and recognized, the cell begins to transcribe CAs genes, thereby producing CAs proteins. what also happens in terms of the CRISPR DNA?

Answer 18

it is transcribed into a long strand of mRNA(including palindromic repeats and spacers)

Question 19

what happens to the transcribed mRNA of the bacterial cell?

Answer 19

it is processed into short guide RNAs called crRNAs each containing a unique spacer sequence that is complimentary to the viral DNA

Question 20

what does the crRNA do once it is made?

Answer 20

makes a complex with cas proteins that neutralize viral DNA when encountered

Question 21

how does the crRNA-cas protein complex work?

Answer 21

when it finds viral DNA, helicase unwinds it, then the crRNA guides the complex to the complementary viral DNA sequence where endonuclease cuts the DNA, neutralizing it

Question 22

what do cas1 and cas2 proteins do the new piece of viral DNA?

Answer 22

incorporate it into the CRISPR sequence as a spacer

Question 23

what are all the elements of the plasmid?

Answer 23

non-coding scrambled RNA sequence, termination signal, CBh promotor(drives Cas9 expression), nuclear localization signal, SpCas9 ribonuclease, another nuclear localization signal, 2A peptide(allows for production of Cas9 and GFP from the same CBh promotor), U6 promotor(drives expression of gRNA)

Question 24

what binds to the unwound viral DNA at the 5’ NGG 3’ spot?

Answer 24

protospacer adjacent motif(PAM)

Question 25

what holds together the hairpin structure of the palindromic repeats?

Answer 25

hydrogen bonds

Question 26

what type of ligation involves the end of the DNA strands being directly ligated back together?

Answer 26

non-homologous end joining (NHEJ)

Question 27

what are the consequences of NHEJ?

Answer 27

often insertions or deletion; used to “knock out” genes

Question 28

what type of ligation involves a DNA template being added to the mix with sequence matching the areas flanking the target site?

Answer 28

homologous recombination (HR)

Question 29

what are the results of HR?

Answer 29

used for precise mutations; “knocks in” genes

Question 30

what protein does CRISPR create to modify the epigenome?

Answer 30

dcas9(dead cas9) which cannot cleave DNA

Question 31

what is the role of gRNA?

Answer 31

GUIDES the cas9 protein to a specific location in the genome to make cuts to the DNA

Question 32

what is gRNA?

Answer 32

short RNA sequence in cas9 that is complementary to a specific target DNA sequence

Question 33

how is gRNA formed?

Answer 33

palindromic repeats(tracrRNA) attach to crRNA

Question 34

what does cas9 recognize?

Answer 34

tracrRNA(palindromic repeats)

Question 35

what does cas9 interact with in CRISPR sequence?

Answer 35

gRNA

Question 36

what must be next to the target DNA for the cas9 complex to bind?

Answer 36

protospacer adjacent motif(PAM)

Question 37

what is the gRNA complementary to if it is made to modify the epigenome?

Answer 37

CpG islands

Question 38

what do gene drives do?

Answer 38

adds the genetic information that codes for the cas9 protein and gRNA that makes up the gene drive to a DNA sequence

Question 39

in an inheritance pattern with gene drives, what is the offspring’s likely genotype?

Answer 39

that of the gene drive, as it is almost always inherited