CRISPR-cas9 and its applications

Question 1

Bacteriophage

Answer 1

A virus that infects prokaryotic organisms.

Question 2

CRISPR-cas9

Answer 2

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 3

Purpose of CRISPR-cas9 in prokaryotes

Answer 3

When a virus hijacks a bacterium’s cell machinery, eventually the virus replicates so much so that the cell lyses and dies. CRISPR-cas9 is their adaptative defence system.

Question 4

Endonuclease

Answer 4

An enzyme that breaks the phosphodiester bond between two nucleotides in a polynucleotide chain

Question 5

Cas9 (CRISPR-associated protein 9)

Answer 5

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

Question 6

CRISPR

Answer 6

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

Question 7

Spacer

Answer 7

Short sequences of DNA obtained from invading bacteriophages that are added to the CRISPR sequence.

Question 8

Protospacer

Answer 8

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

Question 9

PAM (protospacer adjacent motif)

Answer 9

A sequence of 2-6 nucleotides that is found immediately next to the DNA targeted by Cas9.

Question 10

gRNA

Answer 10

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

Question 11

crRNA (CRISPR RNA)

Answer 11

Spacer and a repeat, transcribed and cleaved to produce the copy of viral DNA for Cas-9 to cut DNA.

Question 12

tracrRNA (trans-acting CRISPR RNA)

Answer 12

Complementary sequence to the crRNA repeat which enables the two molecules to bond and establish the final gRNA structure. Also binds tightly with Cas9 to form the complex.

Question 13

Steps of CRISPR-cas9 defence in 3 words

Answer 13

Exposure, expression, extermination

Question 14

Exposure

Answer 14

A bacteriophage injects its DNA into a bacterium, which identifies the viral DNA as foreign. Cas1 and cas2 cut out a short section of viral DNA (typically 30 nucleotides long) known as a protospacer. This can then be introduced into the bacterium’s CRISPR gene and become a spacer.

Question 15

Expression

Answer 15

The CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it, and converted into gRNA. gRNA binds to Cas9 to create a CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that’s complementary to the gRNA. gRNA forms a hairpin loop structure from the transcribed palindromic repeats on either side of the spacer.

Question 16

Extermination

Answer 16

The complex scans the cell for invading bacteriophage DNA that is complementary to the ‘mugshot’ on the gRNA. Cas9 cleaves the sugar-phosphate backbone to inactivate the virus. Cas9 cuts both strands of DNA and create blunt ends.

Question 17

How is the DNA inactivated?

Answer 17

Repair mechanisms in cells are prone to errors that can result in nucleotide additions, deletions or insertions in the middle of the viral gene - rendering the gene non-functional. Otherwise, if the gene is still functional, the process occurs again until repair mechanisms introduce a mutation.

Question 18

Genetic modification

Answer 18

The manipulation of an organism’s genetic material using biotechnology.

Question 19

Deleterious mutation

Answer 19

A change in DNA that negatively affects an individual

Question 20

Gene therapy

Answer 20

Repairing genetic mutations by replacing a defective gene with a healthy one.

Question 21

sgRNA (single guide RNA)

Answer 21

Guide RNA used by scientists to instruct Cas9 to cut a specific site when using CRISPR-Cas9 in gene editing.

Question 22

Gene knockout

Answer 22

Where scientists prevent the expression of a target gene to understand its function in an organism.

Question 23

Using CRISPR-Cas9 in gene editing

Answer 23

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 24

CRISPR-Cas9 and agriculture applications

Answer 24

Improve yield, photosynthetic efficiency, tolerance to environmental conditions, resistance to disease.