It would depend on your system and what you are trying to see.

You can target your shRNA to the 3' UTR of your gene of interest and than reintroduce the mutant without the UTR so it wouldn't be affected.

Using CRISPR you can just introduce the mutation into the endogenous locus so no need to knock it out and reintroduce the mutant somewhere else, this is obviously more complicated to set up than a simple shRNA knockdown but obviously cleaner.

Thanks Yoav for your positive suggestions. 

For the CRISPR directly introduce the mutation, as for as I known the efficiency is pretty low. Since I have 10 mutations to introduce into cells, I wonder if I can knockout the gene by CRISPR, then introduce the mutations by Lenti. The only concern I have is that whether the CRISPR will also cut my introduced gene or not?

The Cas9/gRNA are expressed transiently so by the time you come in with your mutant they should be long gone so even if your gRNA target is still in your construct it wouldn't be affected.

Besides all the above, you can also mutant you exogenous protein that only change the nuclear tide but not the amino acid. Then your exogenous protein will resistant to your siRNA or shRNA which would target endogenous protein.

I am not very sure if the Lenti CRISPR  is expressed transiently or not, since I am planning to make a stable gene KO cell model, and then introduce different mutants.

Thank you, Yanzhou. This would be an alternative approach to achieve mutant introduction without being affect by shRNA.

The cas9/gRNA are usually expressed transiently in order to cut the target sequence out of the genome, once this is done you no longer need them there.

Hi Linzhang,

Can you generated clonal cell line with your cell line of interest?

If yes, I think the best way is to

- Knock down the endogenous gene with a CRISPR/cas9 system which will transiently expresses (with a non integrating lenti for example). 

- Generated and selected the best clonal cell lines which are really knock down for your gene of interest(because with heterogene cell lines, you can have some silent mutation or other strange conformation)

- Introduce all the mutated versions that you want in well characterized cell line with your lenti.

Nicolas

Hi everyone,

I have a question that is related to this topic. Is it possible to create a stable cell line expressing a mutant protein replacing the endogenous one without the use of lentivirus ? 

I have a protein mutated in 2 amino acids and want to replace the endogenous gene by the mutant. Is there any CRISPR or other technique that allow this ?

Thank you !

Vincent

Vincent, you should be able to create your mutant by introducing a homology donor with your desired mutation together with the Cas9 and gRNA.

Thanks for your answer Yoav, is it possible even though the homology donor doesn't have introns ?

The donor should include only the region of your mutation it doesn't need to include all of the gene. If you only want to change a short region it's enough to provide an oligo as donor.

Please have a look at this paper.

Hey Guys,

I wanted to ask a similar question. I want to knockout my gene of interest and introduce a mutant one. But the problem is cells die once they loose the gene. What are my ways to simultaneously do this and see if the mutant rescues my cells? My mutant contains a single mutation, is it more feasible and logical to do Crispr/cas9 to introduce a mutation in the genome? I hope i am clear with my question/

Thanks

I wanted to generate a cell line with point mutation in one of housekeeping gene. This housekeeping gene is related to cell cytoskeleton. So it is impossible to be completely deleted and reintroduce its mutant into the cells. CRISPR/Cas9 knock-in kit might be a good choice, however I have several mutations, it is really time-consuming process. As Yoav Lubelsky @Yoav Lubelsky suggested, what if I knockdown the endogenous gene with the shRNA to UTR and reintroduce its mutant, my concern is that whether we should do it in an opposite way, to introduce the mutant prior to deleting its endogenous gene, and is there any guidance to design shRNA for UTR? Since some genes seems to share the conserved motifs of 3'-UTR. Would somebody give me some guidance or introduction?

Thanks,

Xiaoyan

To knockdown or knockout an endogenous gene of interest and subsequently express its mutants, you can follow a multi-step approach involving gene silencing or disruption followed by the introduction of mutant alleles. Here's a general outline of the process:

By following these steps, you can knockdown or knockout an endogenous gene of interest and subsequently express its mutants to study their functional roles in cellular processes or disease mechanisms. It's important to carefully design and validate your experimental approach to ensure the specificity and efficiency of gene manipulation and the relevance of your findings.

l Take a look at this protocol list; it could assist in understanding and solving the problem.