(Continued) D1.3

Question 1

Gene Knockouts and Conserved Sequences

What is a Gene Knockout?

Answer 1

• Deactive a gene and then monitor the functional loss as a result - involves editing embryos and then targetted mating over a few generations to have organisms that lack that gene in every cell in their body
• Done with MODEL ORGANISMS that have the same gene that is in humans

Question 2

Gene Knockouts and Conserved Sequences

What is a knockout organism?

Answer 2

• An organism that has been genetically engineered to not have a functional gene in any cells in their body
• Also conditional knockout organisms that allows genes to be turned off and on.
• Knockout organisms are then observed for what their functional impairments are based on lacking the gene.

Question 3

Gene Knockouts and Conserved Sequences

What is a Model Organism?

What are four characteristics of them?

Answer 3

• A species that is studied to better understand a human trait.
  1. Short generation times and large offspring numbers
  2. Genome similarity to humans
  3. Small yet well understood genomes
  4. Cost, availability and ethical consideration.

Question 4

Gene Knockouts and Conserved Sequences

What are conserved sequences?

Answer 4

• Identical/nearly identical DNA base sequences across a species/across a group of speices.
• Generally found in protein-coding segments of genome as they’re linked to a specific function that is sensistive to change by mutation

Question 5

Gene Knockouts and Conserved Sequences

What are Highly-Conserved Sequences?

Answer 5

• Remained identical/similar over long periods of evolution, thus are generally seen in highly similar form across a wider range of species.

Question 6

Gene Knockouts and Conserved Sequences

What is the value of model organisms?

Answer 6

• Help to study the impact of specific genes, as other strategies are neither feasible nor ethical to conduct on humans.
• Higher offspring yields give more information - large offspring numbers and short generations are very helpful
• Allow for rigorous and informative genentic experimentation and then the results are then connected back to humans

Question 7

Gene Knockouts and Conserved Sequences

Explain reasearch using knockout mice

Two main reasons

Answer 7

• Libraries of knockout mice that have been made that are lacking a single gene
  1. An MAOA gene (regulates several important neurotransmitters)
  2. TPH2 gene (specifically linked to serotonin
• Both knockouts have been shown to increase aggression in mice.

Question 8

Gene Knockouts and Conserved Sequences

Explain the hypothesis for conserved sequences: Functional Requirements

Answer 8

• One reason some sequences end up being conserved between individuals is that they code for crucial functional proteins
• Wild type sequence codes for a specifically shaped protein associated with high survival & repdocution (highly favoured by natural selection)

Question 9

Gene Knockouts and Conserved Sequences

Explain the hypothesis for conserved sequences: Slower Mutation Rates

Answer 9

• Can also be the result of natural selecton acting against any mutations, and/or it can be the result of existing low rates of mutation due to the DNA being more tightly coiled/ reigons less impacted by mutagens.

Question 10

Crispr Cas-9

What is Gene Editing?

Answer 10

• The intentional and targeted/specific atleration of the genome of an organism
• A search and replace process that identifies an area of interest and uses biotech tools to replace the sequence
• Beneifts: alteration of crops for greater yields, resistance, eradication of human genetic diseases.

Question 11

Crispr Cas-9

What is Cas9?

Answer 11

• Crispr Cas9 is a gene editing tool based on naturally occuring systems in bacter
• Cas9 is an enzyme that uses guide RNA bound to it to find specific sequences in the genome
• It moves along DNA and uncoils it to run along the guide sequence.
• If a target sequence is detected, Cas9 can make a cut to the DNA

Question 12

Crispr Cas-9

What is Guide RNA?

Answer 12

• Attached to the Cas9 enzyme and identifies the specific DNA that needs to be removed or editied.
• When used in biotech, this guide RNA is built to seek out the gene needing editing
• Guide RNA key segment is small and is complementary to the target DNA

Question 13

Crispr Cas-9

What is the Meaning of the C in CRISPR?

Whole term comes from the bacterial immune system

Answer 13

• CLUSTERED: refers to fact that all spacers obtained from previous attacks become stored together close to one anothe rin one strand.
• Meas joined together in one place.

Question 13

Crispr Cas-9

What are PAM Sequences?

Answer 13

• Usually a GG sequence and allows Cas9 to temporarily bind to the DNA, necessary for the endonucleases to cut DNA
• Important for bacteria to ensure they’re only cutting viral DNA and not their own Crispr memory strands.
• Guide RNA must include a complementary sequence to an identified PAM site in the target gene.

Question 14

Crispr Cas-9

What are Endonucleases?

Answer 14

• Specific part of the enzyme that can cut the phosphodiester bond in DNA.
• General term for DNA cutting enzymes, in this context specifically the ability of Cas9 to break a bond on both sides of the DNA requiring two endonuclease regions
• Cut is needed for the repair to occur (how DNA will actually be edited)

Question 15

Crispr Cas-9

What is the Meaning of the R in CRISPR?

Answer 15

• REGULARLY
• Spacer refers to the actual collected viral DNA.
• In between each one are repeats which are bacterial DNA between spacers.
• Repeats are similar in size - creates a very regular pattern in the CRISPR sequence, whereby viral DNA is consistently spread out between repeats.

Question 16

Crispr Cas-9

What is the Meaning of the I in CRISPR?

Answer 16

• INTERSPERSED
• Refers to the fact that there are repeats in between each target RNA spacer.
• They’re not clustered together but instead are in between repeats
• Helps to be identified by Cas9 protein

Question 17

Crispr Cas-9

What is the Meaning of the S in CRISPR?

Answer 17

• SHORT
• Refers to fact that the repeats between the spacer target DNA are only between 23-47 bases long.
• Spacers are unique, can be between 2 and >120 bp in length.

Question 18

Crispr Cas-9

What is the Meaning of the P in CRISPR?

Answer 18

• PALINDROMIC
• As each repeat is a palindrom (something read the same backwards and forwards)
• In Crispr, means the reading of 5’–>3’ would be the same as reading 3’—>5’ backwards
• Causes complementary pairing that creates the loop shape that binds to Cas9

Question 19

Crispr Cas-9

What is the Meaning of the R in CRISPR?

Answer 19

• REPEATS:
• Repeats between the spacer viral DNA that is what is being stored as information
• They’re repeats because the same short sequence appears between spacers
• Repeats form the base of the guide RNA with the spacer then being the end of it.

Question 20

Crispr Cas-9

How can CRISPR Cas-9 be used for gene-editing?

Answer 20

• Guide RNA is created to find the specific sequence to alter
• Then, Cas9 makes a cut and either a base is removed to knock out a gene or an additional sequence is added, or a fault sequence is replaced.

Question 21

Crispr Cas-9

What are the human benefits of Gene editing?

Answer 21

• Could be used to alter crops to address malnutrition or treat genetic orders
• For genetic disorders, would need to be done on embryos
• Can be used to knock out whole chromosomes as a treatment for trisomies
• Can be used to alter mosquitos so they aren’t vectors for malaria, or even to create alternative fuel sources.