lab 10

Question 1

what does CRISPR stand for

Answer 1

clustered regularly interspaced palindromic repeats

Question 2

what system are we studying

Answer 2

CRISPR-Cas9

Question 3

Cas9 enzyme

Answer 3

endonuclease that forms double-stranded break within target site

Question 4

single guide RNA

Answer 4

engineered from of guide RNA that forms a complex with Cas9

Question 5

2 regions of guide RNA

Answer 5

1. guiding - complementary to target DNA sequence, defines where DNA cut
2. scaffold - forms multi hairpin loop that binds in Cas9

Question 6

protospacer adjacent motif

Answer 6

sequence motif downstream of protospacer sequence in recognition sequence that is required for Cas9 function
- allows separation of DNA so sgRNA can bind

Question 7

2 types of DNA repair

Answer 7

1. non-homologous end joining
2. homology directed repair

Question 8

what DNA repair are we using in this lab

Answer 8

homology directed repair

Question 9

what sugar is needed to turn on HDR

Answer 9

arabinose

Question 10

what gene are we targeting

Answer 10

lacZ gene - encodes B-galactosidase

Question 11

2 steps of gene editing

Answer 11

1. cutting dsDNA
2. directing DNA repair

Question 12

what sugar is disrupting by B-galactosidase

Answer 12

X-gal

Question 13

what bacteria are we using

Answer 13

E. coli HB101-pBRKan

Question 14

2 plasmids in this lab

Answer 14

1. pLZDonor
2. pLZDonorGuide

Question 15

pLZDonor

Answer 15

includes donor template DNA, not sgRNA

Question 16

pLZDonorGuide

Answer 16

includes donor template DNA and sgRNA

Question 17

what antibiotic are we suing

Answer 17

spectinomycin

Question 18

protospacer

Answer 18

DNA region targeted for cleavage by the CRISPR system

Question 19

what color does hydrolysis of Xgal by B-galactosidase yield

Answer 19

blue