1-24 Gene Editing

Question 1

Genome editing

Answer 1

group of tech that five scientists ability to change an organisms DNA

add, remove, alter genes

1. targeted DNS strand in IDed 2. target healthy DNA strand is defined and located 3. specifically design synthetic guide molecule finds the target DNA strand 4. enzyme cuts off the target DNA strand 5. target DNA strand is replaced with a healthy one

harnessing natural repair mechanisms to modify DNA:
Need double strand break (DSB):
NHEJ: error prone, just tries to close the gap, indels
Homology directed repair: we can tell cell which template to use to make our change of interest

Question 2

Zinc-finger nucleases

Answer 2

must design a protein to target exact sequence of DNA you want to cut with the nuclease

difficult to design, old version for genome editing

Question 3

TALEN

Answer 3

transcription activator-like effector nucleases- cut DNA at specific spot recognized by the TAL effector domain, found in bacteria but still hard to engineer

Question 4

CRISPR

Answer 4

clustered regularly interspaced short palindromic repeats

nuclease is always the same (Cas9) and you just add in a guide RNA to target the DNA sequence that you want

naturally occurs in bacteria to respond to invading viruses

method: target sequence, you design a guide RNA to complement the target sequence

worry about off-target nuclease activity!

cell may try to repair the CRISPR cut by NHEJ and the outcome is not controllable, you can try to overload the cell with a homologous template to introduce the mutation/change you want to see via ssODNs

Question 5

PAM sequence

Answer 5

NGG located on the uncut DNA strand that the Cas9 associates and helps guide the cutting of the top strand where your guide RNA is designed for

adjacent to the target site

Question 6

ssODNs

Answer 6

single stranded oligodeoxyribonucleotides (SSODNs): short ssDNA can be synthesized and purchased from compaines, up to 200 nt long, used as homologous donor DNA with short homology arms during HR for integration of short stretches of DNA

could enable single nucleotide gene correction for gene therapy purposes or study the phenotypes of diff gene variatns (mice)

Question 7

plasmid double stranded repair template

Answer 7

larger DNA constructs for introducing HR template for gene therapy CRISPR template