L12/13: Nucleic acids - Genomes, CRISPR

Question 1

What is a gene?

Answer 1

• A gene is a sequence of bases in a region of the DNA that can code for a protein or RNA
• Unit of hereditary information consisting of a DNA sequence.

Question 2

Describe the bacterial genome.

Answer 2

• Circular, double-stranded
• Haploid
• all DNA in the cell is tightly wound (supercoiled)
• Genome size (# of base pairs) is small - cells are small

Question 3

Describe the genome in eukaryotes.

Answer 3

• DNA is wound around proteins called histones and wound into chromatin
• Linear, double-stranded
• Diploid

Question 4

What is a genome?

Answer 4

• All the genetic material of a cell/organism

Question 5

What is a genotype?

Answer 5

• The genetic make-up of a cell/organism

- All the genes in the cell

Question 6

What is a phenotype?

Answer 6

• Observable characteristics/traits.

- All the genes that are expressed in the cell due to conditions.

Question 7

Describe the sequence of the composition of the human genome.

Answer 7

• Genomes are complex; contain both coding and non-coding regions of DNA
• only a portion of the human genome codes for proteins (exons, 2.5%)

Question 8

Genome vs. Transcriptome

Answer 8

GENOME:
- all the genetic material in an organism passed from parent to offspring

TRANSCRIPTOME:

• the genes actively being transcribed into mRNA in a given cell, developmental state, etc.
• the entire RNA sequence in a cell.
• the subset of all the genes present in the genome.

Question 9

What does CRISPR stand for?

Answer 9

• Clustered Regularly Interspaced Short Palindromic Repeats

Question 10

What is bacterial immunity?

Answer 10

• Scientists observed that some bacteria could resist and survive a virus attack

Question 11

How was bacterial immunity possible?

Answer 11

• CRISPR DNA

- Some bacteria are able to sample and see DNA from viral genes in special CRISPR arrays.

Question 12

How did CRISPR get discovered?

Answer 12

• Bacteria can get infected by viruses called bacteriophage
• When the cell survives, part of the viral genome is copied in the bacterial genome (i.e short interspaced repeats of sequences of 20-50 bases each)
• Bacterial cell gets infected again, the cell they use RNA (copied from repeats) and a protein Cas9 to edit out the viral DNA

Question 13

What is the difference between spacers and repeats?

Answer 13

• the SPACER is the small section of viral DNA that is captured and inserted into the CRISPR array
• the spacer is always paired up w/ a repeat DNA sequence that is made from the bacterial genome
• the CRISPR array itself is a permanent part of the bacterial genome and can therefore be passed to offspring cells.

Question 14

What are CRISPR arrays?

Answer 14

• Contain all of the viral DNA samples together in a single repeated array
• each spacer sequence is taken from a different virus
• the CRISPR array is a storage bank that records previous encounters with viruses that infect the bacterial cell

Question 15

What is Cas9?

Answer 15

• A protein whose gene is found on another part of the bacterial genome.
• An enzyme that cuts foreign DNA

Question 16

What are the steps in CRISPR?

Answer 16

STEP 1:

• The Cas9 enzymes start scanning all DNA they encounter (including their own) looking for a specific PAM sequence that is unique to viral DNA
• if it finds a PAM sequence, it starts the process of checking the DNA with the crRNA it is carrying.

STEP 2:

• the Cas9 opens the viral DNA and checks it against the crRNA samples it is carrying.
• if it matches, it forms complementary base pairing

STEP 3:
- Once the crRNA and viral DNA are “locked-in”, the Cas9 cuts up the viral DNA (i.e. the Cas9 enzyme is a type of enzyme called a nuclease - cuts nucleic acids)

Question 17

Describe CRISPR and gene editing.

Answer 17

• the crRNA is custom made to match whatever we want to target in the host DNA.
• this type of CRISPR system is also modified in other ways to ensure that the Cas9 recognizes only the target DNA sequence.
• But, there is no guarantee that this won’t happen, which is why most scientists are still wary of testing this out on humans.