Screening! of zebrafish DNA repair mutants generated by CRISPR/Cas9 system - Code : KSI0021

Project : Screening of zebrafish DNA repair mutants generated by CRISPR/Cas9 system

 

The reason why to do this project ? 

- To know the which is the mutant zebrafish and the mutant information  for further study.

 

The necessity of DNA Replication / Recombination / Replication  (3R) mutants on zebrafish
1.Function of DNA 3R processes have been limitedly studied in living organisms.
2.Due to maternal RNA effect with faster development pace, zebrafish mutant embryos can bypass the lethal phenotype compared to mouse model system.
3.Diverse genetic approaches are available in zebrafish system : CRISPR/Cas9.

 

 

Overview of mutant screening process

 

To know or to make the CRISPR gRNA target site > Using BLAT

 

If we know the Forward primer , Reverse primer and Target site sequence, we know the which gene and where is the target exon. Like follows. 

After make the zebrafish mutant, we have to screen the zebrafish is WT or Mutant. 

After genomic DNA extraction > Genetic Analyzer 

This is genetic anaylzer data. 

So we can confirm the 5bp del mutation occured. 

Next we have to further confirm its sequence.

By comparing with WT sequence, we can confirm which sequence is deleted 

And further becuase of that we can identify the framshift. and premature stop codon.

 

This is what I have done in laboratory. 

I learned PCR, BLAT, Sequencing and so on in genomics class. 

I didn't stop by learning the knowledge of genomics but experimented it with critical and independent thinking.

Like ' Is it real? or Is it possible? ' or something like that .

I think there are two big implications here.
One is that I have experimented with what I learned in class and it is because it comes from creative and independent thinking.
Second, these mutant screening processes may seem like nothing, but they contribute to the advancement of science.