Exam 3 Lecture 24

Question 1

Luciferase shortcoming?

Answer 1

No single cell

Question 2

What do bacteria use for adaptive immunity?

Answer 2

CRISPR/Cas

Question 3

crRNA

Answer 3

RNA made from the DNA from an invader that was inserted into the CRISPR array

Used as a guide to find/match to the invader if they invade again

Question 4

What is the structure of the CRISPR/Cas gene complex?

Answer 4

Cas genes which are Type I, II or III systems then an AT-rich leader

Then come the 27-37 bp palindromic repeats separated by 32-38 bp unique sequences

Question 5

Cas genes first job?

Answer 5

Aquisition or adaptation

Cas1 (binds) and 2 (cleaves) grab invading DNA and PAM sequence next to it and insert it into the CRISPER array in between repeats

Question 6

CRISPR/Cas Type I function?

Answer 6

Simpler

crRNA’s made from genes that were inserted aka their memory

Cas proteins bind crRNA ~35 bps and match it to invading DNA sequence next to a PAM site and cleave the invader

Kill!!!

Question 7

How big are PAM sites and explain their function?

Answer 7

3-5 bps, they are a set sequence according to the Cas gene.

The same Cas gene will always pick the same 3-5 bps every time and take the DNA sequence just upstream from them. That way when they are invaded again they match the PAM which they know from their innate memory and the DNA sequence they grabbed which they remember using the crRNA made from the CRISPR array.

Question 8

Are the invading gene sequences inserted in the CRISPR array in any order?

Answer 8

Yes in chronological order as the bacteria encounters them

Question 9

Type II difference?

Answer 9

They have a tracr gene upstream from the Cas genes that is used to help process the CRISPR genes and bind to them during use making a CRISPR Cas9/crRNA/tracr complex that binds and cleaves the foreign DNA

The 5’ end of the tracr RNA bind to the crRNA

Question 10

Modification to Cas9 to use it in gene modification?

Answer 10

Instead of a tracr/crRNA Cas complex they can combine the two separate tracr and crRNA’s into one chimera guide RNA

Question 11

What happens after the gene is cut?

Answer 11

1. NHEJ or Non-Homologous End Joining (happens with Crispr only)
2. Alternative Allele is provided and added into cut

Question 12

Steps of NHEJ/Non-homologous end joining?

Answer 12

1. Ku protein binds each end
2. A combination of a polymerase, exonuclease, and Ligase called LigD is recruited by Ku.
3. LigD Polishes and ligates the ends together

Question 13

Problem with NHEJ?

Answer 13

Error prone

Usually, a few nucleotides added = frameshift mutations

Question 14

Another use for a catalytically dead Cas9 (dCas9)?

Answer 14

Sequence-specific repression of gene transcription- it will bind and block the initiation site so RNA poly can’t bind.

CRISPR Interference