Prediction of PAM sequences
All CRISPR arrays were retrieved from CRISPRCasdb, part of CRISPR-Cas++, which can be found at https://crisprcas.i2bc.paris-saclay.fr/ (28). Alignment of CRISPR array spacers to genomes was done via BLAST (29) either programmatically using Spacer2PAM or manually through the web interface. The BL...
Chemically modified oligonucleotide synthesis
Standard phosphoramidite solid-phase synthesis conditions were used for the synthesis of all modified and unmodified oligonucleotides. Syntheses were performed on an Applied Biosystems 3400 or Expedite DNA Synthesizer at a 1 micromole scale using Unylink CPG support (ChemGenes)...
Do you have any recommendations in terms of tools for guide RNA design for cas13? And does the crRNA of active cas13 works for deactivated cas13d.
I would like to delete/mutate a regulatory element in the promoter of a gene in HEK 293T cell lines. However, I have never tried designing guides/donors for knock in/HDR.
Can some one recommend me a protocol and vectors/design strategies for this?
What are the easiest/most efficient strategies for this at the moment?
I am using Gecko v2 library to screen survival cells after drug treatments.
After drug treatments, I have harvested cells and count the cell numbers.
As expected, I have 4 times more numbers of cells in the control group (untreated) than in tested groups. I expect that I have more genomic DNA in the control group too.
So...
I m approaching to the CRISPR/Cas9 world. I m going to perform an experiment using CRISPR/Cas9 mediated knockouts in primary human T cells from donor PBMC cells and I have some doubts regarding the method.
I found two different approaches:
the CRISPR/Cas9- mediated gene editing that requires the endonuclease Cas9 and a gu...
is there any specific guide RNA software tool for labeled genomes ( like fluorescent protein sequences) ?
Can lentiSAMv2 (with a guide RNA) activate without using lentiMPH v2? I recall that lentiMPH v2 just improves the efficiency of activation for some genes.
I did a gene activation experiment 6 months ago in fibroblasts where I used a guide RNA known to work in HEK293 cells. The guide also worked well in all my experiments using fibroblasts (nucleofection, Lonza Amaxa kit). I am now trying to repeat this overexpression experiment in the same fibroblast cell line but I am not se...

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