4B - Crispr Cas 9

Question 1

CRISPR-Cas9 

Answer 1

a complex formed between gRNA and Cas9
- can cut a target sequence of DNA.

Bacteria use - protection from viruses and scientists have modified it to edit genomes

Question 2

(CRISPR)

Answer 2

Clustered Regularly
Interspaced Short Palindromic Repeats

Question 3

(Cas9)

Answer 3

CRISPR-associated protein 9
an endonuclease that
creates a blunt end cut at a site specified by guide RNA

Question 4

spacer 

Answer 4

short sequences of
DNA obtained from invading bacteriophages that are added into
the CRISPR sequence

Question 5

protospacer adjacent motif (PAM) 

Answer 5

a sequence of two-six nucleotides
that is found immediately next to the DNA targeted by Cas9

Question 6

guide RNA (gRNA) 

Answer 6

RNA which has a specific
sequence determined by CRISPR to guide Cas9 to a specific site

Question 7

What are the three steps of the CRISPR-Cas9 system in bacteria?

Answer 7

Exposure – bacteriophage (virus) infects a bacterium, the bacterium captures a small piece of the viral DNA (protospacer) and stores it in its CRISPR region as a “mugshot.”

Expression –virus invades again - bacterium transcribes the stored viral DNA into guide RNA (gRNA), which then binds to the Cas9 enzyme.

Extermination – The gRNA guides Cas9 to the matching viral DNA, allowing Cas9 to cut (blunt ends) and destroy the virus, preventing infection.

Question 8

crispr cas 9 for gene editing

Answer 8

1. Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA
   that scientists wish to cut.

2 A Cas9 enzyme is obtained with an appropriate target PAM sequence.

3 Cas9 and sgRNA are added together in a mixture and bind together to create the
CRISPR-Cas9 complex.

4 The sgRNA-Cas9 mixture is then injected into a specific cell, such as a zygote.

5 The Cas9 finds the target PAM sequence and checks whether the sgRNA aligns with
the DNA.

6 Cas9 cuts the selected sequence of DNA.

7 The DNA has a blunt end cut that the cell will attempt to repair.

8 When repairing the DNA, the cell may introduce new nucleotides into the DNA at this
site. Scientists may inject particular nucleotide sequences into the cell with the hope
that it will ligate into the gap

Question 9

purpose of crispr cas 9 in prokaryotes

Answer 9

CRISPR-Cas9 cuts target DNA, according to the complementary sequence provided by gRNA, to combat bacteriophage invasion.

Question 10

grna

Answer 10

guide rna
directs cas 9 to target dna squence