Genetic and molecular approaches: genetic screens

Question 1

Identification of genes controlling development(Genetic analysis)

Answer 1

• Genetic analysis
  
  • Mutagenesis screens-random mutagenesis and selection by phenotypic analysis
  • Reverse genetics-mutation of specific DNA sequences and analysing the phenotype
• Transgenics

Question 2

Identification of genes controlling development(Molecular methods)

Answer 2

Methods to identify genes with restricted expression patterns in the embryo and genetic interactions

Question 3

How do we genetically screen for recessive mutations in zebrafish and how do we identify the gene?

Answer 3

• ENU mutagenesis
• Keep F1 males
• Cross F2 animals
• If F2 cross is between 2 carriers, 25% of their offspring will be homozygotes for the mutation
• Establish line
• Identify gene by
• Complementation analysis
• Genetic mapping
• Positional cloning

Question 4

How do you identify phenotypic outcome?

Answer 4

• Morphological defects
• Changes in other genes expression
• Changes in the expression of a transgene highlighting a particular tissue/cell type

Question 5

What are 2 editing tools to target sites for mutation?

Answer 5

• ZFNs and TALENs

- CRISPR-Cas9

Question 6

What are ZFNs?

Answer 6

Zinc finger nucleases

Question 7

What are TALENS?

Answer 7

Transcription activator like effector nucleases

Question 8

What do ZFNs and TALENS recognise?

Answer 8

-These proteins recognise specific DNA sequences, attached to an endonuclease domain

Question 9

What do endonucleases cause?

Answer 9

-Endonucleases cause a cut activating the repair machinery and imprecisely fixes the bases causing a mutation

Question 10

What does CRISPR stand for?

Answer 10

-Clustered interspersed short palindromic repairs

Question 11

What is CRISPR-Cas9 based on?

Answer 11

-Based on bacterial adaptive immune system

Question 12

What is DNA recognition in CRISPR-9?

Answer 12

-DNA recognition based in a guide RNA with a protospacer adjacent Motif

Question 13

How does the CRISPR-Cas9 system work?

Answer 13

• We have a sgRNA(single guided RNA) molecule which is made up a target specific crRNA sequence and a tracrRNA region
• TracrRNA allows to become assembled with the Cas9 protein
• Cas9 has endonuclease properties
• The sgRNA+cas9 protein complex targets specific cleavage
• Then the repair mechanism in the cell will imprecisely prepare generating a mutation in that region