lecture 18 - recombinant dna tech 5

Question 1

what is genome editing

Answer 1

Changing in sequence of DNA (change bases, adding tags, deletics suppresssors, anything that changes the thing)

Question 2

what were early engineered nucleases

Answer 2

Transcription Activator-like Effector Nucleases (TALENs)
Zinc Finger Nucleases (ZFNs)

Question 3

how do TALENs work

Answer 3

Have dual recognition sites
Can cleaves double stranded DNA

Question 4

how do ZFNs work

Answer 4

Composed of zinc finger domains to recognize DNA
Each finger recognize between 9 and 18 bases

Question 5

what does CRISPR stand for

Answer 5

clustered regularly interspaced short palindromic repeats

Question 6

where is CRISPR originally found

Answer 6

bacteria

Question 7

what does CRISPR locus have

Answer 7

CAS genes, and repeat sequences with spacers (spacers are variable DNA)

Question 8

what is CRISPR purpose in bacteria/archaea species

Answer 8

works as bacteria immune system (the memory for how it has been affected)

Dna sequence from virus is derived and integrated into CRISPR sequence which when formed as RNA can direct the molecular machinery to target virus so they can kill it

Question 9

what are the components of Cas9 system and what do they do

Answer 9

• Cas 9 protein → cleaves the DNA
  [Has HNH and RuvC domains (each cleaves a different strand)]
• CRISPR has RNA sequence that matches up with DNA you want to cut
• tracrRNA connected Cas9 and CRISPR to help with leading
• can optionally have repair template which is ssDNA

Question 10

what is snRNA

Answer 10

guide RNA which a combo of crRNA with tracrRNA

Question 11

what is the PAM sequence in CRISPR

Answer 11

piece of foreign DNA (phage) lies upstread of PAM sequence to help with directing (Pam sequence is NGG in Type II systems)

Question 12

what are limitations to CRISPR

Answer 12

• PAM sequence can limit where you can have editing (bigger issue for knock ins)
• Crispr has possibility of cutting genome at nontargeted sites throught the genome (low frequency but still matters for health care)
• Two component system – need more sgRNA and the Cas9 protein

Question 13

what are applications of CRISPR Cas 9

Answer 13

• gene knockout
• genetic knock in
• editing base pair
• non editing application
• injecting zygotes
• correcting genetic diseases

Question 14

what are some things you need to consider for gene knockout

Answer 14

Mutations cannot be multiple of 3 or have to be very carefully screened to know if this will change a protein and have it still work

Question 15

how can crispr cas 9 edit base pairs

Answer 15

Use cas9 to direct another protein to a place in the system and then can do cytosine to uracil through cytidine deamination (mismatch reapri preseves edit if modified strand is used as template)

Question 16

what are non editing applications of crispr cas 9

Answer 16

• Can artificially activate gene of interest by having dead cas9 that has a tag on it get brought to a promoter – no double stranded break here
• Can do this with a repressor as well
• These can also been directed to silencers and something else not just promoters
• Can use cas9 to tag with fluorescent colors
• can create pooled crispr screens (useful for reverse genetics screens for phenotype of interest)

Question 17

how are cataracts being treated for mice

Answer 17

using CRISPR to repair mutation in crygc gene that causes cataracts

Question 18

how is Progeria being treated for mice

Answer 18

issue is single base change in LMNA gene that results in 150 bp shorted mRNA, can use crispr to restore the exon 11 sequence and fix this protein

Question 19

what are some limits in treating diseases with CRISPR

Answer 19

• Off target edits
• Delivery methods → how do you get the complex to the right type of cells in patients. Actively being worked on
• Editing efficiency → unlikely to work on 100% of cells in patients
• DSB breaks can cause problems in cells

Question 20

what is the main crispr system

Answer 20

type II