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 

Enhancing sgRNA Expression in Mammalian Cells


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 advance for your assistance.
Best regards,
Anan 

dCasRx/13 and splicing 

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 transposase. While I understand that it detects transcription factors and cuts accordingly, I am seeking more information on the length of the resulting fragments and whether this aspect can be controlled.
If anyone has experience using the CUT&Tag Kit from Active Motif and could provide me with answers to these questions, I would greatly appreciate it.

Thank you in advance for your assistance!

Best regards, 

Anan

CUT & TAG Kit - Tn5

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 

CRISPR ARRAY - Cas13 

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 experience in addressing this matter and can offer guidance, or knows of any specific cloning techniques that may be useful, I would greatly appreciate your assistance. (e.g The NLS should be at C/N-Terminal?, The NLS should be followed by HA/HIS Tag? What about stop codon? )

I would highly appreciate any help or advice on this topic.

Best regards,

 Anan

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