Gene editing

Question 1

name gene editing techniques?

Answer 1

1. zinc finger nucleases (ZFNs)
2. transcription activator like effector nucleases (TALENs)
3. CRISPR

Question 2

how do gene editing techniques work?

Answer 2

DOUBLE STRAND BREAK at nuclease target site

cause…
gene insertion
gene correction
gene disruption

Question 3

how does gene insertion/correction occur?

Answer 3

template recognises n aligns where the double stranded break occurred
DNA donor template aligns and inserts new gene and undergoes homologous directed repair

Question 4

how does gene disruption occur?

Answer 4

two DNA ends join = non-homologous end-joining = gene disruption

Question 5

how do ZFNs work?

Answer 5

use a specific site to induce DNA-DS break - using 3 BASE PAIRS UNITS

Binds to both sides of the DNA-DS

Question 6

how do TALENs work?

Answer 6

same as ZFNs however increase sensitivity
recognises ONE BASE PAIR UNIT

Question 7

what is CRISPR-cas 9?

Answer 7

cas genes
CRISPR - Clustered regularly interspaced short palindromic repeats

Question 8

How is CRISPR-cas 9 used?

Answer 8

in drug discovery - target identification/validation

Question 9

describe CRISPR/Cas-9 positive screening?

Answer 9

• Introduce Cas9 and guide RNA for targeted gene mutation
• Cas9 cuts DNA at the target gene
• Donor DNA introduces modifies DNA with a fluorescent marker
• Cells use Donor DNA for repair
• Repair mechanism include NHEJ and HRR
• Cells with successful modifications express the fluorescent marker
• Choose cells with the fluorescent marker for further studying

Question 10

describe CRISPR/Cas-9 screening?

Answer 10

It involves designing a guide RNA (gRNA) that directs the Cas-9 protein to a specific gene, inducing a break in the DNA.

Cellular repair mechanisms then lead to gene disruption or precise editing.

The process requires screening to identify cells with the desired edits using techniques like PCR and DNA sequencing.

Question 11

describe CRISPR/Cas-9 negative screening?

Answer 11

Cells that haven’t incorporated the desired genetic modifaction will not exhibit the fluroescence associated with the modifed DNA.

By excluding these non-edited cell, researchers can narrow down their focus to the cells that have successfully undergone the intended geentic changes.

Negtaive Screening is a way to filter out unwanted outcomes and enhance the efficiency to identifying edited cells in CRISPR/Cas9.