Hi,
I have a CRISPR system based on two AAV vectors 1/2, the titers are 10^12 for the gRNA and 10^10 for the Cas9. I'm having troubles in the proccess of primary neurons transduction - I can't infect the cells as planned (the gRNA have a EGFP marker, and the expression rate as measured in FACS sorter is around 3-5% of all cells).
Can anyone help me and share their transduction protocol?
How much viral solution should I add to a 6-well neuron culture? in what volume should I do the transduction? how long after the seeding of the primary neurons in the well should I transfect the cells with the virus? for how long should I leave the virus in the cell culture before renewing the media? any tips are welcome!
HI Shani,
Did you try non integrating lentiviral vectors? This type of vector is very useful to transduce your type of cells and their high cargo capacity allow to optimize the delivery of CRISPR system. We use them in my lab, including in neurons, throught a two vector system and also a all in one system allowing to have one event of transduction bringing the two CRISPR elements.
Regards
Nicolas
Hi Julia and Nicolas, thank you for your answers.
We store the virus in -80 temp, and we used Lenti in the past but it didn't work well in-vivo, so we switched to AAV
Ok, -80 is good.
In vivo, the lenti can less diffuse than AAV, however, poor results can also due to a low efficiency of the LVs. In vivo, we use lenti with a titer between 1E9-1E10 TU/mL and have good results. In my experience, lenti with a titer
Hi Shani
AAV isn’t great for in vitro delivery. You may want to follow advice of Nicolas Grandchamp and use LV to validate your CRISPR in vitro and then switch to AAV for in vivo work.
But if you want to get the most out of AAV in vitro, try a high dose. I use doses ranging from MOI 10,000 to MOI 100,000 for AAV in vitro. The MOI is basically the ratio of virus genomes to human genomes. So for example, if you have 1,000,000 cells in the 6 well plate, this means you would need 10^10 to 10^11 AAV genomes to get the necessary MOI. That would require ~10 to 100 microlitres of your gRNA AAV, but it also requires around 1-10 ml of your Cas9 prep...so you’ll probably need a better titre for the Cas9 vector. Or, you could experiment with a reduced dose range of the Cas9 vector, eg try MOI 1000, 5000, 10000, to see whether these doses work.
In terms of other questions, you can transduce cells with the AAV immediately after plating in 1ml media (for 6 well plates) and it’s best to leave the vector in the media for at least 24 hours. It’s not really necessary to do a media change - fine to leave it in.
All the best
I know with some viruses you can get a dramatic reduction in titer with every freeze-thaw cycle, so just be aware of that.
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