SAC 1? - CRISPR-Cas9

Question 1

What type of enzyme is Cas9?

Answer 1

Crispr - Associated = Cas9
- endonuclease in bacteria

Question 2

What is CRISPR and where is it found?

Answer 2

• Clustered Regularly Interspaced Short Palindromic Repeats.
• naturally occurring sequences of DNA that plays an important role in defence against viral attacks
• found in bacteria

Question 3

how CRISPR -Cas9 acts as a defence system in bacteria

Answer 3

1. Viral DNA enters the bacteria and protospacer is removed by endonuclease
2. protospacer is inserted into the CRISPR region as a spacer between repeats, providing memory
3. upon reinfection, CRISPR gene is transcribed, each CRISPR spacer is transcribed into guide RNA (gRNA).
4. The gRNA forms a complex with the Cas9 enzyme (CRISPR-Cas9 complex).
5. The gRNA guides the complex to a complementary viral DNA sequence.
6. Cas9 cuts the viral DNA, inactivating the virus and stopping it from replicating.

Question 4

Role of Cas 9

Answer 4

• endonuclease creating blunt ends
• acts as ‘molecular scissors’
• guided to the right DNA sequence to cut by gRNA
• Coded for by the Cas9 gene upstream of CRISPR sequence

Question 5

What is a bacteriophage?

Answer 5

a virus that infects prokaryotes

Question 6

What are protospacers

Answer 6

a short sequence of DNA, made BEFORE a spacer

Question 7

what are spacers

Answer 7

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

Question 8

What is the CRISPR gene made of?

Answer 8

• clustered REPEARS of nucleotides, interrupted by SPACER DNA

Question 9

repeats vs spacers

Answer 9

repeats - Short, identical DNA sequences that repeat multiple times
spacers - Unique DNA sequences between the repeats, each taken from past viruses.

Question 10

What is gRNA?

Answer 10

(guide RNA) - a short RNA sequence made from the CRISPR region that includes a copy of a viral spacer

Question 11

what is the role of gRNA?

Answer 11

• guies Cas 9 to a complementary DNA sequence in the virus to cut

Question 12

What role does the CRISPR-Cas9 complex play with viral reinfection?

Answer 12

• upon reinfection, CRISPR genes are transcribed into gRNA
• piece of gRNA forms a CRISPR-Cas9 complex with Cas9
• gRNA directs the CRISPR-Cas9 complex to the complementary sequence
• Cas9 cuts both strands of DNA, inactivating the virus and preventing it from replicating

Question 13

Why is the PAM sequence important to Cas 9 function?

Answer 13

• increases efficiency of Cas9 in finding target DNA when looking upstream of viral DNA
• protects bacteria - Cas9 only cuts DNA if there is a PAM next to the target sequence, and the bacteria’s CRISPR DNA does not have PAMs next to the spacers, so Cas9 won’t cut its own DNA.

Question 14

Explain the steps in using CRISPR-Cas9 to edit genomes.

Answer 14

• sgRNA is created, complementary to the target gene
• CRISPR-Cas9 complex created, and injected into target cell
• cas9 cuts DNA strands forming blunt ends, after being guided
• enzymes try and REPAIR the break, but this process is prone to errors
• this can induce mutations OR nucleotides being added that are incorporated = stops/changes gene’s function

Question 15

Gene knock-in

Answer 15

• adding a gene to an organisms geneome, introducing foreign DNA from other organisms

uses: 1) introduce a favourable allele in agriculture ie. frost resistance
2) research a gene in a model organism

Question 16

Gene knock-out

Answer 16

• Scientists design piece of sgRNA that is complementary to target se
• the silencing of a gene by altering its nucleotide sequence
• so it cannot be transcribed/translated into a protein
  = can also determine the function of a gene in model organisms
  = can also introduce a particular mutation

Question 17

State some applications (in medicine) of using the CRISPR-Cas9 system for gene editing.

Answer 17

medicine - GENE THERAPY - dealing with diseases ie. huntington’s, cystic fibrosis.
- replacing deleterious allele with healthy
- adding genes that code for proteins to decrease susceptibility to infectious diseases
- modifying cancer-promoting genes to make them less influential

Question 18

State some applications (in agriculture) of using the CRISPR-Cas9 system for gene editing.

Answer 18

• improving nutritional value = increasing amount of nutrients in crops
• enhancing disease resistance = protection from viruses, fungi, + other pests
• altering genes to promote increases growth rates to improve yield of crops

Question 19

issue of non-maleficence in the use of CRISPR-Cas9

Answer 19

• opposing genetically modified embryos due to unforeseen negative consequences on the pregnancy and child later in life
  OR
  What if the use of CRISPR-Cas9 reduces the pain and suffering of child that had a genetic condition?

Question 20

issue of beneficence in the use of CRISPR-Cas9

Answer 20

• using CRISPR-Cas9 to improve health ie. curing genetic diseases
• making sure edits are safe, effective, and truly benefit individuals, without causing harm

Question 21

issue of integrity in the use of CRISPR-Cas9

Answer 21

• sourcing + referencing info honestly
• reporting true results (favourable and unfavourable)
• avoid data manipulation, and follow ethical guidelines

Question 22

issue of respect in the use of CRISPR-Cas9

Answer 22

• welfare, liberty, automony, beliefs, customs, and cultural heritage should be considered
• ensuring people give informed consent for gene editing, and their privacy, dignity, and beliefs are respected

Question 23

issue of justice in the use of CRISPR-Cas9

Answer 23

• competing claims to be addressed and considered
• no unfair burden of a particular group
• fair access/distribution of benefits and equal treament
• gene editing should not only be available to the rich/certain groups