Gene editing

Question 1

What are ZFNs?

Answer 1

Zinc finger domains that dimerise to cut DNA

Question 2

What 2 ways do ZFNs cut DNA?

Answer 2

NHEJ- non-homologous end joining- short indels disrupt genes for knockout

HR if a template sequence is in introduced for knockins

Question 3

Describe the first ZFN transgenic rat

Answer 3

Targeted immunoglobulin heavy chain μ locus (IgM)

Knockout to study the role of antibodies

Question 4

What 2 components are needed for CRISPR?

Answer 4

A synthetic target-specific guide RNA (gRNA)

CAS9 endonuclease

Question 5

What is in the gRNA?

Answer 5

tracrRNA with a target-specific crRNA sequence and a ds region that guides CAS9 into position

Question 6

What does CAS9 do?

Answer 6

Recognises and binds to a 3-9bp proto-spacer adjacent motif (PAM) and then unwinds the ds chromosomal DNA and the complementary region of the gRNA hybridises which triggers CAS9 to change in 3D structure and activates its nuclease activity to cut the DNA upstream of PAM

Question 7

Describe gene editing in cattle

Answer 7

Knockout myostatin gene which inhibits muscle development using TALENS using injection into egg cytoplasm for larger cattle

Question 8

What are some unintended effects of CRISPR?

Answer 8

Off-target effects in similar sequences
Mosaicism and multiple alleles
Integration of template vectors when creating knockins