Enhancing sgRNA Expression in Mammalian Cells
Hi,
I'm working with sgRNA expression in mammalian cells using the U6 promoter. Are there stronger promoters or modifications to increase sgRNA or CRISPR array expression?
Thanks,
Anan
dCasRx/13 and splicing
dCasRx/13 and splicing
I am reaching out to inquire if anyone in the community has experience using dCasRx to target exons or introns, specifically in the context of observing changes in splicing patterns. If you have worked with dCasRx in this manner, I would be grateful if you could share your insights or any relevant resources.
Thank you in ad...
CUT & TAG Kit - Tn5
CUT & TAG Kit - Tn5
Greetings everyone!
I am interested in using CUT&TAG to pull down transcription factors and have some questions about the cutting range of the technique. Specifically, I'm curious if the Tn5 transposase employed in CUT&TAG can cut RNA or if it only cuts DNA. Additionally, I'd like to know the cutting range of the Tn5 transp...
CRISPR ARRAY - Cas13
CRISPR ARRAY - Cas13
Hello!
Has anyone successfully performed Gibson assembly for six Cas13 guides? If so, did you incorporate the direct repeat sequence within your ordered guides?
I know how to do it to Cas9 sgRNAs but a little bit confusing for Cas13 because of the repetitive DR.
Any help will be highly appreciated.
Best,
Anan
Cloning SV40-NLS and HA Tag to dCas9 expressed plasmid
Cloning SV40-NLS and HA Tag to dCas9 expressed plasmid
Greetings everyone,
I currently have a dCas9 backbone that lacks an NLS signal necessary for nuclear expression, as well as any tag for conducting Pulldown assays. While I am aware that I can obtain a plasmid with the necessary NLS and Tag, I am interested in exploring alternative solutions.
If anyone has previous experie...
DNA yield lower than predicted, how to proceed?
DNA yield lower than predicted, how to proceed?
I'm isolating DNA from frozen cell pellets at 2e7 for a half-library screen using HEK293T cells.
My issue is that from each 2e7 pellet I am obtaining approximately 45-60 ug, while given a 6 pg/cell estimation I should have ~110-120ug.
I am fairly certain that the cell count is correct, as all cells were re-seeded to flask...
CRISPR gene deletion
CRISPR gene deletion
I want to delete a gene using a Transient CRISPR method. I have few queries regarding gene deletion:
1\. I want to use single gRNA with high off and on target score. How efficient it could be or I can use 2 gRNAs for better efficiency.
2\. I want to use 3 stop codons followed by restriction site and marker gene. Is there ...
question about SAM
question about SAM
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 h...
Virus Titration befor infection with Gecko Library
Virus Titration befor infection with Gecko Library
I'm going to use gecko library in order to infect primary macrophages and is the very first time I'm approaching this technique, so I have some questions.
I'm following the nat prot paper by Julia Joung (2017) but I have some questions regarding the titration part.
I'm using the 2 vectors system and I will infect macropha...
Silent promoter activation with dCas9-activators - opinions
Silent promoter activation with dCas9-activators - opinions
What do you think, why can be a promoter being transcriptionally silent? If it is silent, do to that there is no native transcriptional activator binding to this promoter, could this be supplemented with a well-guided dCas9-activator?