Gene Editing

Question 1

What natural repair mechanisms does gene editing take advantage of?

Answer 1

Double stranded break repair

• homology directed repair
• NHEJ

Question 2

Zinc finger nucleases

Answer 2

• Engineered proteins (DNA cutting endonuclease fused to Zn finger domain engineered to bind a specific DNA sequence) that bind and cut specific DNA sequences
• Indels at cut sites in living cells
• 10% success

Question 3

Transcription activator-like effector nucleases

Answer 3

• TAL effectors are secreted DNA-binding proteins from bacteria that are engineered to recognize specific sequences
• Indels at cut sites in living cells
• Increased specificity over ZFNs

Question 4

Naturally occurring bacterial CRISPR

Answer 4

Used as a bacterial immune system:

1. After viral attack, bacteria collect pieces of viral DNA and incorporate them into their own genome at the CRISPR locus (cas genes + repeats and spacers)
2. These fragments are transcribed, and next time the virus attacks, the crRNAs guide cas proteins to the virus to cleave DNA

Question 5

Cas9

Answer 5

Enzyme that makes a double stranded break

Question 6

gRNA

Answer 6

20 nt sequence complementary to the target attached to a longer RNA scaffold

Question 7

PAM sequence

Answer 7

• Protospacer adjacent motif
• 3 nt sequence needed for Cas activity (different for each Cas)
• Must occur in the genome next to the target region and increases specificity

Question 8

What is the success rate of CRISPR with homologous recombination?

Answer 8

< 10%

Question 9

What are the sources of template DNA for homologous recombination in CRISPR?

Answer 9

plasmids and ssODNs

Question 10

ssODNs

Answer 10

• single-stranded oligodeoxyribonucleotides
• used for single nucleotide gene correction
• 200 nt fragments with short homology arms

Question 11

plasmids (for use in CRISPR)

Answer 11

• 800-1400bp regions of homology flanking site of interest

Question 12

Germline therapies

Answer 12

change DNA in reproductive cells; changes will be passed on

Question 13

Somatic therapies

Answer 13

changes will only affect that individual