question about SAM

chenyang zheng

unread,

Apr 1, 2021, 5:21:19 AM4/1/21

to Genome Engineering using CRISPR/Cas Systems

Dear all:

We recently prepared to use SAM for screening experiments, but we found that the lentivirus titer of the package was always very low, and the cells were almost dead after adding puromycin. In addition, another CRISPRa library we used (Calabrese, from john doench lab) also had such a problem. Other lentiviruses we packaged have no problem, such as LentiCRISPR V2. We are very confused. Does anyone know the reason?

BTW, the cell we used is HepG2. In the preliminary experiment to measure the infection rate, we were not infected with dCas9-vp64 and MPH.

THANKS.

BEST WISHES

ZCY

Julia Joung

unread,

Apr 5, 2021, 11:38:35 AM4/5/21

to chenyang zheng, Genome Engineering using CRISPR/Cas Systems

Hi ZCY,

This is the first time I have heard of this problem. In A375 and HUES66 cells, I did not see any differences in lentivirus titer between CRISPR activation and CRISPR knockout. Could you maybe test the lentiviral titer in HEK293 cells or a different cell line to make sure this is not cell line dependent?

Best,

Julia

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msgid/crispr/23373595-e049-4f6f-8a4b-6c7145bc908bn%40googlegroups.com.

Rike

unread,

Sep 13, 2021, 4:37:01 AM9/13/21

to Genome Engineering using CRISPR/Cas Systems

Hey ZCY,

not sure if that helps ypu right now, but I am encountering exactly the same problem - with both SAM and Calabrese. GeCKO and TKOv3 were no problem.

While I do get some functional viral particles, the titer is very low (around 2e5 TU/ml in my desired cell line) and very cell line dependent higher in H1299, OKish in HEK293T).

If you found a way on how to improve the titer I'd be extremely grateful if you could share your protocol/hacks :) I'm currently using FuGene6 as transfection reagent.

Best,

Rike

Hamish McWilliam

unread,

Sep 14, 2021, 6:36:13 PM9/14/21

to Rike, Genome Engineering using CRISPR/Cas Systems

Hi Rike and ZCY

I’m also having problems with viral titres (with Calabrese).

If anyone has got some tips for us then please send in.

Best

Hamish

On 13 Sep 2021, at 6:37 pm, 'Rike' via Genome Engineering using CRISPR/Cas Systems <cri...@googlegroups.com> wrote:

Hey ZCY,

To view this discussion on the web visit https://groups.google.com/d/msgid/crispr/8c84f8f3-41ca-42ef-80d0-4e03828e9b83n%40googlegroups.com.

Reply all

Reply to author

Forward