How do I make my own mutant? Part 2

Question 1

Pathogens and innate immunity

Answer 1

• blocks entry

- blocks attachment to the host cell

Question 2

Pathogens and adaptive immunity

Answer 2

• injection of pathogenic DNA/RNA into host cell
• integration into host DNA
• CRISPR
• recognition
• cleave incoming pathogenic DNA/RNA

Question 3

What is the CRISPR-Cas system?

Answer 3

• occur naturally in bacteria and archaea

- protect these organisms against bacteriophages plasmids and other invading DNA elements

Question 4

What is crRNA?

Answer 4

• CRISPR RNA

- encoded by DNA sequences called Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)

Question 5

What is CRISPR?

Answer 5

• consist of palindromic sequences separated by unique spacers
• derived fro bacteriophages or foreign plasmids
• proteins cut up the foreign DNA and insert bits of it into a CRISPR array
• now serves a memory of the invader
• the CRISPR array is transcribed into a long precursor CRISPR RNA (pre-crRNA)
• is cleaved into short crRNA

Question 6

What are Cas proteins?

Answer 6

• combines with crRNAs to form effector complexes
• have nuclease activity
• the ability to cut DNA

Question 7

What are the effector complexes?

Answer 7

• when the same foreign DNA enters cell in the future
• CRISPR-Cas complex recognizes and attaches to it
• crRNA binds to its complementary sequences in the foreign DNA
• Cas protein cleaves foreign DNA
• now non-functional

Question 8

What are the PAM sequences?

Answer 8

• protospacer associated motifs
• short sequences with weak consequences
• so occurs at numerous random places in the genome
• CRISPR-cas9 effector complex first associates with
• does not exist in bacterial genomes

Question 9

What is guide RNA?

Answer 9

• a specific RNA sequence that recognizes the target DNA region of interest
• made up of 2 parts
• crRNA and tracrRNA
• tracrRNA serves as a binding scaffold for the Cas nuclease

Question 10

What is the role of non-homologous end-joining?

Answer 10

• joins together 2 ends of DNA without a template
• introduces small insertions and deletions when the two ends are joined
• allows geneticists to disable a gene because of frameshift mutations

Question 11

What is the role of homologous recombination?

Answer 11

• when a DNA template is provided to repair db breaks
• can provide a donor piece of db DNA with ends complementary to the sequences at the ends of breaks made by Cas9
• researchers can selectively insert the desired sequence into a genome
• not highly efficient
• often the 2 ends are connected without insertion of donor DNA

Question 12

What is CRISPRi?

Answer 12

• modified Cas9 that can’t cut
• blocks elongation
• modified Cas9 can’t cut attached to GFP
• modified Cas9 can’t cut attached to an activator
• increases expression
• modified Cas9 can’t cut attached to a suppressor
• decrease expression

Question 13

What are some CRISPR-Cas9 uses?

Answer 13

• so far worked in every organism
• simple system
• knock out, knock-in or alter genes
• adjust gene expression
• potential use for gene therapy
• can disrupt just mutated alleles

Question 14

What are some CRISPR-Cas9 limits?

Answer 14

• off-target effects
• can handle mismatch of 1-5 nucleotides
• new versions and methods are addressing this
• in adult humans, getting it to intended cells

Question 15

Relationship with gene drives and inheritance

Answer 15

• normal inheritance
• altered gene does not spread
• gene drive inheritance
• altered gene is always inherited

Question 16

What is CRISPR mediated gene drive?

Answer 16

• a self-spreading CRISPR system
• one homolog has insert sequence that produces Cas9, gRNA and the gene of interest
• a germline started heterozygous for “gene X”
• CRISPR is transcribed and translated and cuts gRNA target site
• repairs homologous recombination
• now homozygous for “gene X”