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In general, if there are multiple genes of interests, or edits at multiple points along a large gene are desired, can several edits done at once (or as many as is possible while minimizing off-target effects), transfecting cells with multiple gRNAs and possibly different Cas variants, or would the edits be done one at a time, with each edit being screened for one after another? Or somewhere in-between where you'd batch edits and screen for them in groups? Mostly interested in base edits and electroporation based transfection, but any information on workflow for editing multiple sites is welcome!

Per bob1, my goal is to edit multiple base pairs on sites adjacent to and within a gene for in vitro mammalian cell lines, to see how those edits affect protein expression.

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    $\begingroup$ How you go about it is dependent on what phenomena you are interested in and your reasons for looking at the products. Need more information on your aims to answer this. Also depends on organism. $\endgroup$
    – bob1
    Sep 26, 2023 at 19:18
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    $\begingroup$ The workflow you're talking about I believe is called multiplexing. Maybe this article can help you out. $\endgroup$ Sep 26, 2023 at 19:50
  • $\begingroup$ @pawelekzzz That's exactly the term I'm looking for, thanks! I didn't know the name of it and hadn't seen that term in any of my searches prior, this gives me what I need the find more articles, and the one you link gives a good intro to the concept too, much appreciated. $\endgroup$
    – Gumpf
    Sep 26, 2023 at 20:28
  • $\begingroup$ @bob1 Simultaneous base pair edits on sites, upstream, downstream and mid-gene for in vitro mammalian cell lines, to monitor changes in protein expression $\endgroup$
    – Gumpf
    Sep 26, 2023 at 20:32
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    $\begingroup$ @pawelekzzz you could expand that into an answer. $\endgroup$
    – bob1
    Sep 26, 2023 at 20:42

2 Answers 2

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One of the advantages of CRISPR/Cas9 is that it allows what is called multiplexing. In other words: transcriptional regulation of multiple different genomic loci in one single construct. I'm not sure if other biotechnological methods allow for a different strategy to obtain the same result.

Multiplexing using CRISPR/Cas9 has been documented several times expressing several gRNAs at once. Anecdotally, I can also personally say it works. The multiplexed CRISPR/Cas9 reagents are assembled into T-DNA vectors. This is done through reactions called Golden Gate cloning and Gateway reaction.

Bear in mind I'm talking about plants all this time.


References:

Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X, Voytas DF, Hsieh TF, Zhang Y, Qi Y. A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiol. 2015 Oct;169(2):971-85. doi: 10.1104/pp.15.00636. Epub 2015 Aug 21. PMID: 26297141; PMCID: PMC4587453.

Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013 Aug;31(8):688-91. doi: 10.1038/nbt.2654. PMID: 23929339; PMCID: PMC4078740.

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What you should do is highly dependent on what you are looking at and why you want to make those changes. It also depends on which method you want to use

There are quite a few different ways of doing this for mammalian cells. One commonly used way is to have the gene expressed off a plasmid in a cell line that lacks expression of the protein of interest. This means you have an easily manipulable plasmid which you can make and confirm sequence changes easily and quickly. Plasmids are also easy to use and can get results quickly. However, these are usually non-permanent changes, unless you have a selection mechanism like geneticin and maintain this on your cells. If you don't maintain the selection the cells tend to lose the plasmid. The downside is that you don't tend to get every cell transfected and the percent of cells transfected drop with plasmid size and/or method and can be variable between different transfections and different plasmid preparations. These methods are known as transient transfection and stable transfection respectively. For many sequential changes, looking at intermediates, this is probably the method I would use, but CRISPR/Cas (see below) is also promising.

For targeting of the genome of the cells you are looking at, conventional gene editing techniques by recombination between plasmid and genome are slow and best suited to smaller inserts and low numbers. They tend to have lower success rates and require things like antibiotic selection to ensure that they have happened. This includes things like Cre-Lox recombination. These tend to be quite slow relative to transient transfections and require you to have robust sequence checking methods in place (often done by PCR product size and/or sequencing), but are stable and don't require continued selection once stable. They can be done at a single-cell level and can be site-specific, depending on the method used.

Viral transduction is similar to both transfection and recombination methods. You usually work with relatively easily manipulated virus genomes expressed off plasmids to make the viruses, which are then used to do the recombination/expression. It does tend to have quite a bit of finicky set-up, but is robust and stable once it is working. Lentivirus and Adenoviral vectors are two common methods of this, but there are others.

CRISPR/Cas I don't know a lot about, but I know it is entirely possible to use this method to rapidly make site specific changes in genomes. I think this is the most promising technique for many genetic manipulations in the cell. This is very quick, but requires quite a bit of optimization.

In general, making lots of changes at once is entirely possible, though, as you will have realized from the above, this depends on the mechanism used to make the changes. Just be aware, that if you are manipulating the genome of a host, dramatic changes (i.e. many) might drastically affect the cells in some fundamental manner so that you don't get any cells survive. This is where intermediate steps might be useful, but is something you can choose or not at this point.

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    $\begingroup$ Really? :) You provided the animal cells answer, and I provided the, rather succinct, plant cell version. $\endgroup$ Sep 26, 2023 at 21:15
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    $\begingroup$ @pawelekzzz To be fair, you can use the viral method and a bacterial method (Agrobacterium i think?) on plant cells too. $\endgroup$
    – bob1
    Sep 26, 2023 at 21:40
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    $\begingroup$ Yes, that's right, Agrobacterium tumefaciens is used in plant cells transformation. $\endgroup$ Sep 26, 2023 at 21:42
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    $\begingroup$ @pawelekzzz excellent, having not worked on plants for over 20 years, good to know my memory of methods hasn't failed on me. Not that I used Agrobacterium at the time, just knew it was a possibility. $\endgroup$
    – bob1
    Sep 26, 2023 at 21:48

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