Help please with CRISPR by transient transfection of LentiGuide-Puro

[Jemma Milburn]

Hello,

I am attempting a slightly odd CRISPR protocol and wanted to run it by some experts please.

I have transduced HEK293T cells using LentiCas9-Blast ( #52962) Because I am going to be doing 5 rounds of CRISPR and FACS on them in total. (As an aside, they are not very stable, I am still getting cell death even after 3 weeks of Blasticidin selection (I chose the lowest conc’ on my kill curve). I will consider making colonies, but any links to protocols here would help please.)

I now want to do CRISPR gene knock-out by transient transfection only, of my guides in LentiGuide-Puro (#52963.) So far I haven’t had success.

Will the guide RNA definitely be expressed following transient transfection? I realise the plasmid is 3^rd gen and has it’s own promoter – so far so good – but will the RNA be synthesised from the correct strand?

My reason for doing this is two fold. I need the guides to be lost, and hope that passaging after transient transfection will be enough to do this.  I need to KO the endogenous gene, but I am then transducing a different haplotype of the same gene, and I don’t want the CRISPR to get to work on that. Also, the guide vector has PuroR, and I need to transduce with something else, later on with PuroR, so I also need the resistance to be lost (using a different antibiotic wasn’t an option).

I also don’t think I need to rely on Puro selection for the CRISPR because conveniently I can FACS for cells that have lost surface expression of the target.

My plan is to try again, with this loose protocol: Seed 6 well plates with Basticidin resistant HEK (in antibiotic free media) to 70% confluency. Transfect for 6-hrs using PEI and 3 X Lenti-G vectors which target three conserved regions across 3 genes). Change media, split when needed. Add puro selection 72 hrs later to half (to test if its necessary/helpful). Flow sort 4-5 days post transfection.

Is this plan hopeless and should I fork out for ribonucleoprotein complex, or do I have some chance of success?

Many thanks

Jemma