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There are multiple widely-used plasmids for using CRISPR/Cas9 with a dual lentiviral vector strategy (Cas9 & sgRNA on different vectors) in mammalian hosts with a Blasticidin selection selection marker.

In particular: Lenti‐Cas9‐2A‐Blast(#73310) and lentiCas9-Blast (#52962)

What are differences between the two? Which vector is recommended (for use in mouse small intestinal organoids) and why?

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  • $\begingroup$ You will have to include a lot more details about your protocol in order for anyone to give you pros and cons abot those two vectors. What kind of mammalian host? Please edit your question accordingly. If there's a protocol in your lab, just use that one and see if it works. But I recommend that you do your own research and learn about the purpose of each and every entry of both plasmid-charts, so you know what you're doing. $\endgroup$
    – markur
    Sep 7, 2022 at 13:25
  • $\begingroup$ I tried to to ask the question in way that the answer is useful for many people, but I included the host now. $\endgroup$
    – jan-glx
    Sep 16, 2022 at 12:25
  • $\begingroup$ It might also help if you add the type of host cells that produce your lentivirus. $\endgroup$
    – markur
    Sep 16, 2022 at 13:17

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I see these differences in lenti-Cas9-2A (compared to lenti-Cas9):

  • Absence of a bleomycin resistance. That just affects cloning
  • Changing from a CMV- to a RSV promoter for lentiviral mRNA production: That promoter is used for the host cells that produce your lentiviruses. They might react differently to different promotoers. So in the end, your viral titers might differ, when switching these plasmids.
  • Changing from a bGH- to a SV40 polyA signal polyA signal. That might affect (production and) half-life of lentiviral mRNA both in your lentivirus producing-host cell culture and your mouse small intestinal organoids after transfection. You might want to start your research here: Humes et al. 2020

Together, I think both plasmids should work just fine. I'd say that the observed differences affect mostly how well your host cell culture performs in producing lentivirus. And these difference might differ a lot between cell types. But like I said, it's just the lentivirus production. If you got enough virus to apply to your mouse organoids for gene editing, it doesn't matter.

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