Block D Workshop - Applications of CRISPR / Cas

Question 1

What does CRISPR stand for?

Answer 1

Clustered Regularly Interspaced Short Palindromic Repeats
(Slide 5)

Question 2

What percentage of sequenced bacteria and archaeal genomes is CRISPR present in?

Answer 2

~50% of bacterial and ~90% of archaeal
(Slide 5)

Question 3

What do CRISPR-Cas systems help protect organisms from?

Answer 3

Foreign DNA (such as bacteriophages and plasmids)
(Slide 5)

Question 4

What is the anatomy of a CRISPR loci?

Answer 4

DNA repeats each separated by a spacer sequence
(Slide 6)

Question 5

What are the 3 stages of CRISPR activity?

Answer 5

Adaptation
Expression
Interference
(Slide 7)

Question 6

What occurs in the adaptation stage of CRISPR activity?

Answer 6

The prokaryotic organism encounters foreign genetic material, then cas proteins capture short segments of this foreign DNA and incorporate them into the CRISPR array as new spacer sequences
(Slide 7)

Question 7

What does the adaptation stage of CRISPR activity require?

Answer 7

Cas ribonucleases or RNase III
(Slide 7)

Question 8

What occurs in the expression stage of CRISPR activity?

Answer 8

Transcription + processing of the CRISPR RNA (crRNAs) with the crRNAs and cas proteins assembling to form CRISPR ribonucleoprotein (crRNP)
(Slide 7)

Question 9

What occurs in the interference stage of CRISPR activity?

Answer 9

crRNP (CRISPR Ribonucleoprotein) scans DNA for target sequences resulting in target degradation
(Slide 7)

Question 10

What are 3 applications of CRISPR / Cas)?

Answer 10

Genome editing
Gene silencing
Diagnostics (via detecting of specific nucleic acid sequences)
(Slide 11)

Question 11

How can CRISPR/ Cas 9 be used for genome editing?

Answer 11

Use an RNA (crRNA + tracrRNA or sgRNA) to target a genome sequence of interest
(Slide 12)

Question 12

What must the genome sequence of interest in CRISPR genome editing be adjacent to?

Answer 12

A PAM (protospacer adjacent motif) on the non-target DNA strand
(Slide 12)

Question 13

What occurs after CRISPR cuts the DNA in genome editing?

Answer 13

The resulting DNA double-strand break can be repaired by the native DNA repair pathway

Question 14

What occurs in the nickases variation of CRISPR genome editing?

Answer 14

Target strand / non-target strand cleaved only, depending on mutation
(Slide 17)

Question 15

What occurs in the dCas9 variation of CRISPR genome editing?

Answer 15

It is catalytically dead and therefore has no ability to cleave DNA
(Slide 17)

Question 16

What is the first step of CRISPR interference (CRISPRi)?

Answer 16

Catalytically inactive dCas9 is targeted to a gene of interest (usually at or very near the promoter)
(Slide 18)

Question 17

What occurs in CRISPR interference (CRISPRi) after dCas9 is targeted to a gene of interest?

Answer 17

dCas9 binds and represses transcription, resulting in no expression of the target gene
(Slide 18)

Question 18

How can CRISPR induce expression of a particular gene(s)?

Answer 18

By targeting dCas9 to the promoter in such a way that you will activate expression
(Slide 19)

Question 19

What 2 ways can dCas9 be expressed?

Answer 19

It can be constitutively expressed or inducible
(Slide 19)