delete N-terminal domain from protein of interest

[regulatingge...@gmail.com]

Hi,
I want to delete the first 50 amino acid from my protein of interest (ie: delete 150 bp) and maintain the orf of the rest of the protein.
I have 2 options:
1. use 2 gRNAs that flank the sequence to be deleted. The problem is that a lot of the indels can cause out of frame deletions

2. use HDR by cotransfecting 1 gRNA and a rescue vector that has homology arms complementary to the sequences before and after the deletion. I do not know how efficient the HDR in comparison to NHEJ?

Does any one have suggestions which option to use? Thanks.

[didier Fesquet]

You can insert your deleted protein  construct in aavs1 locus in a endogenius homozygous  deleted background. Many possibilities

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+unsubscribe@googlegroups.com.
To post to this group, send email to cri...@googlegroups.com.
For more options, visit https://groups.google.com/d/optout.

[regulatingge...@gmail.com]

Thanks. Sorry I meant I want to delete the N-terminus from the endogenous locus instead of deleting the gene and then reinserting truncated orf in AAVS, is there any way to do this efficiently?
Thanks.

On Thursday, February 16, 2017 at 1:18:59 AM UTC-6, didier Fesquet wrote:

You can insert your deleted protein  construct in aavs1 locus in a endogenius homozygous  deleted background. Many possibilities

Le 15 févr. 2017 02:00, <regulatingge...@gmail.com> a écrit :

Hi,
I want to delete the first 50 amino acid from my protein of interest (ie: delete 150 bp) and maintain the orf of the rest of the protein.
I have 2 options:
1. use 2 gRNAs that flank the sequence to be deleted. The problem is that a lot of the indels can cause out of frame deletions

2. use HDR by cotransfecting 1 gRNA and a rescue vector that has homology arms complementary to the sequences before and after the deletion. I do not know how efficient the HDR in comparison to NHEJ?

Does any one have suggestions which option to use? Thanks.

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.

To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.

[Marine C]

Hi!

In which cell type do you want to do it?

The option by HDR is the best in your case because you will have clean repair, but the HDR efficiency depends on different things : cell type, gRNA efficiency, donor design..
Do you think that you can use a donor with a selection marker? Maybe with loxP site around the marker.. but you will have a scar at the end, maybe it's not a problem in your case.
Any way, with a selection marker it should be easy to find the right clone. Because even if you have a high rate of HDR, it will be really hard to find a clone with every gene copies modified.

[regulatingge...@gmail.com]

Hi,
My cell line is RPE1 because they do not have polyploidy
Is 1000 nucleotides good enough for homology?
Would it be easier if I stick GFP ( and sort using FACS) than using a drug resistance marker, because I do not want the drug resistance gene to be translated at the N terminus.

[Marine C]

Ok! RPE1 are not known to have a high rate of HDR, so it's really a good idea to use a marker to enrich your positive cells.
You can use GFP yes, but won't it be a problem after? Just like with drug resistance?
Or you can use a Cas9 plasmid containing GFP or a Cas9 fused to GFP... So you can sort the cells with transfected Cas9 and enrich your postive cells. It won't be as good as if you can insert GFP byhomology but it can be a good solution.