Exercise question

[Roundwaytrip]

My professor gave me some assignments, sort of like homework and I'm struggling with one and I hope you can help me.

Let's say I'm working with an iPSC line that expresses a fluorescent mCherry tag which is fused to the histone H2B protein. I use CRISPR/Cas9 to knock out several target genes and after that I need to generate control iPSC lines which are genetically identical but don't express the mCherry fluorescence. So, what would the strategy for this be, what are the potential challenges and risks and how to verify the successful disruption?

 

[ClintonClay2]

You'd use CRISPR to knock out or remove the mCherry tag, isolate clones, and verify by sequencing and fluorescence loss, but you have to watch for off-target effects and ensure H2B stays functional

 

[Caitlyn P]

My professor gave me some assignments, sort of like homework and I'm struggling with one and I hope you can help me.

Let's say I'm working with an iPSC line that expresses a fluorescent mCherry tag which is fused to the histone H2B protein. I use CRISPR/Cas9 to knock out several target genes and after that I need to generate control iPSC lines which are genetically identical but don't express the mCherry fluorescence. So, what would the strategy for this be, what are the potential challenges and risks and how to verify the successful disruption?

@ClintonClay2 is right and biggest issues are off target effects or messing with expression. To check if it worked, see if the red signal has gone under a scope, then confirm with PCR or sequencing
.

 

[Roundwaytrip]

@ClintonClay2 is right and biggest issues are off target effects or messing with expression. To check if it worked, see if the red signal has gone under a scope, then confirm with PCR or sequencing
.

Can CRISPR introduce mutations elsewhere in the genome?

 

[crisprnavigator25]

You'd use CRISPR to knock out or remove the mCherry tag, isolate clones, and verify by sequencing and fluorescence loss, but you have to watch for off-target effects and ensure H2B stays functional

Okay, so CRISPR knockout does the trick, but keep an eye out for those off-targets and double-check the H2B function.