Hello William.

It depends on the property of the target gene. If your GOI is a transmembrane receptor or the truncated region is an active domain interacting with other crucial proteins, then the strategy of truncation mutant would work to find any phenotypic changes. Otherwise it would not.

There is also issues in which the GOI should work uniquely without overlapping with other signaling pathways. Which case you are wondering 'Is this specific to my mutation or combined effects from other sharing molecules?' Probe with siRNA to knock-down the GOI to see the effects first, and then decide the feasibility.

Regards.

Yes i think CRISPR would be the best way. You could design CRISPR plasmids to introduce stop codons at several places in the gene to see where the critical binding/interations occur within the protein?

Hello William,

if the KO is truly lethal than your approach is the only possible one. CRISPR works just great in 293 and you will not have problems in isolating KO clones. In order to produce the KO I would rather use two sgRNAs flanking the whole CDS (or at least a substantial part of it) in order to create a deletion.

Use of single sgRNAs for KO can induce protein rearrangement downstream your targeted sequence and restart translation from alternative ATG or re-arrange the splicing isoforms to produce N-terminally truncated, non physiological proteins.

This is a summary of the steps I would follow:

I would first produce a KI of your wt CDS in your cell line, flanked by two frt/flp sequences and a selection cassette between them (let's say Puro). You can KI this transgene in a safe harbour (Rosa26 for instance) using CRISPR and HDR.

You can also use T-rex Flip-in cells for this purpose.

This transgene will provide a backup copy of your protein for your KO. Once you establish this cell line you can proceed with knocking out the endogenous copy (by targeting again with CRISPR a couple of region outside the cDNA in the 5' and 3' UTR for instance).

Now you can select for KO clones and pick 2-3 with homozygous deletion for consistency. At this point your cells will still be viable thanks to the transgene you've previously integrated in the safe-harbour.

At this point all you have to do is to generate your truncated constructs flanked by frt/flp sequences and co/transfect them in your engineered cell line together with a Flippase encoding plasmid. The wt CDS you've inserted in the safe harbour will be exchanged for the truncated version you are providing, enabling control over copy number and assessment of the phenotype in an isogenic cell line (coming from a single KO event).

You can have your truncated construct with Hygromycin for selection (your mutants should have lost Puromycin and gained Hygromycin resistance).

If you think this protocol is too long (I think it would take 3-6 months at least) , you can substitute safe/harbour integration of your wtCDS with lentiviruses encoding both wt and truncated proteins but that would mean that you will first have to establish the overexpressing cell-lines and then remove the endogenous protein in each one of them by CRISPR/Cas9 (which is an hard work and will potentially generate genetically different KO models which could alter your results). Overall the two approaches have the same difficulties and length I believe.

Hope it helps and good luck

Francesco

Thank you all for the answers.

We have performed knockdown of the GOI with RNAi and have shown the effects in cells. We also already know the C-terminus of the protein can still remain functional even without the N-terminus, however this was never tested in cells.

Thank you Francesco for the details, but is the step of KI of wt CDS (the back up step) necessary? The plan I originally had was to produce a KI of the gene for the truncated protein and if the cells showed similar phenotypes as the OE of wt CDS as well as not being lethal, I would proceed to KO the endogenous gene.

Hi William,

it is not strictly necessary but it would be nice as a control. Some exogenous promoter are weaker then the endogenous one (depending on the protein you are studying) and some effects that you will attribute to the mutant protein might as well be because of differences in protein expression levels.

If you have also a wt KI you can use it as a control.

Hope it helps

Francesco

Hi Francesco

Thank you very much again for the answer.