bio 4B

Question 1

what is the ‘exposure’ process of CRISPR-Cas9

Answer 1

1. bacteriophage injects its DNA into bacteria and the bacteria identifies it as a foreign substance, where enzymes cut out a small segment of the bacteriophage DNA (protospacer) and introduced into the DNA of the bacteria as a part of the CRISPR gene (spacer)

Question 1

what is CRISPR-Cas9

Answer 1

a section of DNA with short, repeated sequences on nucleotides that are interrupted by a spacer DNA and is an endonuclease that creates a blunt end cut at a site specified by guide RNA (gRNA)

Question 2

what is the ‘expression’ process of CRISPR-Cas9

Answer 2

CRISPR spaces are transcribed along with half a palindrome from the repeat on either side of it and is converted to an RNA molecule (gRNA) where gRNA then binds to cas9, forming a hairpin loop

Question 3

what is the ‘termination’ process of CRISPR-Cas9

Answer 3

the CRISPR-Cas9 complex scans the cell for invading bacteriophage DNA (complementary to the gRNA inside itself) and when it finds some, cleaves the phosphodiester bond of the sugar-backbone to inactivate the virus. because of this, the viral gene will want to repair itself and the blunt end cut can mean that errors can occur in this stage (addition/deletion of nucleotides) which can render the bacteriophage non-functional. the CRISPR-Cas9 complex continues this until the mutation occurs

Question 4

what is gene therapy

Answer 4

it refers to repairing genetic mutations by removing the defective gene with a healthy one (is rarely used outside of clinical trials as it can inadvertently insert genes into the wrong part of the genome)

Question 5

what is sgRNA (single guide RNA) and its role

Answer 5

it is made up of one RNA molecule rather than two RNA molecules and is guide RNA utilised by scientists to instruct Cas9 to cut a specific site when using CRISPR-Cas9 in gene editing

Question 6

steps to use CRISPR-Cas9 for gene editing

Answer 6

1. synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA that is to be cut
2. Cas9 sequence is acquired with an appropriate target PAM sequence
3. Cas9 and CRISPR are added together and form the CRISPR-Cas9 complex and is inserted into the cell
4. Cas9 targets the specific sequence and checks if the sgRNA aligns with the DNA
5. Cas9 cuts the sequence of DNA
6. DNA now has blunt ends and attempts to repair itself
7. while repairing, mutations to the DNA may occur

Question 7

what are limitations of CRISPR-Cas9

Answer 7

• scientists can not yet use it on humans to eliminate genetic diseases
• it is difficult to induce substitution mutations or knock-in a new statement of DNA with precision and be consistently successful (as scientists must add the nucleotide sequence and only hope that it is taken up by the DNA repair machinery)
• ethical implications of using CRISPR-Cas9 (e.g. scientists must treat an embryo prior to differentiation to ensure all DNA is altered - this can hit the concern of the respect fo the sanctity of life)

Question 8

what is a PAM sequence

Answer 8

a sequence of 2-5 nucleotides found immediately next to the DNA targeted by cas9

Question 9

what is Cas9

Answer 9

an endonuclease that cuts the double-stranded DNA helix

Question 10

what is CRISPR

Answer 10

short, clustered repeats of DNA found in prokaryotes which protect them against viral invasion

Question 11

what is gRNA

Answer 11

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

Question 12

what is a gene knockout

Answer 12

a technique in gene editing where scientists
prevent the expression of a target gene to understand its function in an organism

Question 13

what is a gene knock-in

Answer 13

a technique in gene editing where scientists substitute or add nucleotides in a gene