4B Crispr CAS-9

Question 1

whats a virus

Answer 1

a non-cellular, infectious agent composed of genetic material enclosed in a protein coat that requires a host cell to multiply

Question 2

whats bacteriaphage

Answer 2

a virus that infects prokaryotic organisms

Question 3

whats CRISPR-Cas 9

Answer 3

a complex formed between gRNA and Cas9 which can cut a target sequence of DNA. Bacteria use this complex for protection from viruses and scientists have modified it to edit genomes

Question 4

whats CRISPR-associated protein 9 (Cas9)

Answer 4

an endonuclease that creates a blunt end cut at a site specified by guide RNA (gRNA)

Question 5

whats a spacer

Answer 5

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

Question 6

how does CRISPR work

Answer 6

A bacterium takes a portion of DNA from a viral infection and incorporating it into their own genome. Next time it is infected by the same type of virus the bacterium transcribes the ‘mugshot’ DNA and attaches it to an endonuclease called Cas9.
The transcribed mugshot is complementary to the viral DNA, so it ensures that the Cas9 only destroys the invading virus rather than any bacterial nucleic acids.
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats
The clustered repeats are interrupted by spacer DNA, which is the viral ‘mugshot’.
CRISPR sequences are always downstream of the gene for Cas9.

Question 7

whats a protospacer

Answer 7

Protospacer: a short sequence of DNA extracted from a bacteriophage by Cas1 and Cas2, which has yet to be incorporated into the CRISPR gene

Question 8

whats Protospacer adjacent motif (PAM):

Answer 8

a sequence of 2-6 nucleotides that is found immediately next to the DNA targeted by Cas9. Required for Cas9 to bind to the DNA.

Question 9

whats guide RNA (gRNA)

Answer 9

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

Question 10

what are the 3 steps of how CRISPR works

Answer 10

When a bacterium is infected by a bacteriophage, there are three steps to fighting the virus with the CRISPR-Cas9 system:
Exposure
Expression
Extermination.

Question 11

PAM sequence

Answer 11

The protospacer adjacent motif (PAM) isa short DNA sequence (usually 2-6 base pairs in length) that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9. The PAM is required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site.

Question 12

whats step 1 of how CRISPR works

Answer 12

Exposure
The bacteriophage injects its DNA into a bacterium, which identifies the viral DNA as a foreign substance.
Cas1 and Cas2 (CRISPR-associated enzymes like Cas9) enzymes cut out a short section of the viral DNA (typically ~30 nucleotides long), known as a protospacer.
This protospacer can then be introduced into the bacterium’s CRISPR gene and become a spacer.

Question 13

whats step 2 of how CRISPR works

Answer 13

Expression
The CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it, and converted into an RNA molecule known as guide RNA (gRNA).
gRNA binds to Cas9 to create a CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that is complementary to the gRNA
gRNA forms a hairpin loop-like structure from the transcribed palindromic repeats either side of the spacer.

Question 14

whats step 3 of how CRISPR works

Answer 14

Extermination
The CRISPR-Cas9 complex then scans the cell for invading bacteriophage DNA that is complementary to the ‘mugshot’ sequence on the gRNA.
When it does, Cas9 cleaves the phosphate-sugar backbone to inactivate the virus.
Cas9 contains two active sites to cut both strands of DNA and create blunt ends.

Question 15

what happens when the viral DNA is cut

Answer 15

When the viral DNA is cut, enzymes within the bacterium will naturally act to repair it.
The repair mechanisms in a cell are prone to errors that can result in nucleotide additions, deletions, or insertions in the middle of the viral gene.
This is advantageous in the case of bacteriophage infiltration because these mutations tend to render viral genes non-functional.
If a mutation does not occur after the cut, the gRNA will find the gene again and repeat the whole process until the DNA repair mechanisms induce a mutation, inactivating the virus.

Question 16

whats genetic modfication

Answer 16

the manipulation of an organism’s genetic material using biotechnology

Question 17

whats gene therapy

Answer 17

repairing genetic mutations by replacing a defective gene with a healthy one

Question 18

whats gene knockout

Answer 18

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

Question 19

whats gene knock in

Answer 19

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

Question 20

how can CRISPR be used for gene editing

Answer 20

The CRISPR-Cas9 system can induce genetic changes by cutting DNA at a location specifically chosen by scientists, who make a synthetic gRNA to guide Cas9. This creates an opportunity for nucleotides to be added, removed, or substituted into the selected sequence. In turn, this can knockout, enhance, or change the function of a gene.

Question 21

how to use CRISPR-Cas 9 for gene editing

Answer 21

CRISPR-Cas9 is not the only mechanism available but it is currently the most precise and affordable option.
To use CRISPR-Cas9 for gene editing, the following steps must be taken:
1. Synthetic gRNA 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 gRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex.
4. The gRNA-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 gRNA 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 22

whats non homologous end joining (NHEJ

Answer 22

When a double strand break (DSB) occurs, organisms utilise non-homologous end joining to repair itself without needing a template strand. This can be from random breaks or from accidental cuts from endonucleases. Around 10-50 DSB occurs everyday.
When a DSB is sensed, enzymes are brought to the site of the break. The ends are brought together and DNA ligase creates phosphate-sugar bonds between the nucleotides.
NHEJ is not perfect, sometimes nucleotides are added or lost. This makes the process error prone.
Scientists can use this error prone system to induce mutations in the DNA that can ‘knock-out’ a gene.
‘Knock-out’ refers to the gene’s inability to function. The gene is still there, just non-functional

Question 23

whats homology directed repair (HDR)

Answer 23

Another process in which DSB are repaired is through the use of homologous DNA. These will have the same nucleotides as the broken strand, and can be used as a template to repair the DSB.
There are two main processes: Synthesis Dependent Strand Annealing (SDSA) or Double Strand Break Repair (DSBR)

Question 24

whats synthesis dependent strand annealing (SDSA)

Answer 24

Resection occurs to cut away some of the nucleotides on one strand of the DNA until it gets to a Chi site (GCTGGTGG).
The 3’ end will stick out and get dragged by another enzyme to the homologous section (D loop) through complementary base pairing
DNA polymerase copies and extend the 3’ end until it reaches the chi site. The attraction between the original strand will become stronger than the homologous strand so it will be brought back to it’s original strand
DNA ligase will ligate them together

Question 25

whats double strand break repair (DSBR)

Answer 25

Same as SDSA
Same as SDSA but instead of only the 3’, both 3’ and 5’ ends are pulled into the D loop.
DNA polymerase extends till the 5’ chi site
Repair occurs at both ends at the same time but due to both ends being in the loop, crossing over of the strands can occur

Question 26

limitations in CRISPR Cas 9

Answer 26

While tested in mice there is potential use in humans but genetic modification of humans is outlawed in many countries.
CRISPR-Cas9 simply cuts DNA at a chosen site. To induce substitution mutations or knock-in a new segment of DNA, scientists must introduce the nucleotide sequence they wish to add into the cell and hope it is taken up by the DNA repair machinery which is difficult imprecise and not consistently successful.
Progress has been slow is because of the ethical implications of developing effective CRISPR-Cas9 technologies

Question 27

problems with CRISPR Cas 9 bioethics

Answer 27

Safety – The possibility of off-target cleavages (edits in the wrong place) and mosaics (some cells containing edited genomes, others not) mean that many scientists are hesitant to use CRISPR outside of research.
Informed consent – Scientists cannot get consent from embryos to edit their genes. If the embryo goes on to be born and one day has children of its own, these children also will never have consented to scientists interfering with their genome.
Inequality – There is concern that only wealthy people will be able to afford to use CRISPR to treat genetic conditions or otherwise change their genes.
Discrimination – CRISPR may be a threat to those who are judged by society as biologically inferior, when in fact those individuals do not feel they need ‘fixing’ at all.