Gene knockout protocol from HCC cancer cell lines using px459-v2?
I would be highly appreciated if someone could share a detailed protocol (worked) for knocking out genes from any HCC cancer cell lines using px459-v2.
Thank you
Cas13 RNA design for knockdown of chicken cells
Cas13 RNA design for knockdown of chicken cells
I have experiences in RNAi and Cas9 knockout experiments in mammalian cells, but am now facing a little challenge.
I have primary chicken and duck endothelial cells that require knockdowns of genes - knockouts of these genes are impossible due to their importance for these cells.
I have seen 1-2 papers looking at CMV/Ef1a...
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...
Stable Cas9 expressing hESCs H1
Stable Cas9 expressing hESCs H1
I am in the process of developing a CRISPR screen which requires Cas9 expressing hESCs. I have been following the protocol outlined in the Joung et al. 2017 Nature protocols paper. However, I am having a lot of difficulty producing a Cas9 expressing H1 cell line. I have transduced these cells in parallel with a GFP lentivir...
dCas9-VP64 Lenti System
dCas9-VP64 Lenti System
Apologies for what is probably a simple question. I'm essentially hoping to mentally generate a user guide for dCas9 () akin to the PDF for the LentiCRISPR protocol, but for the dCas9 system.
Am I right that to target a gene for gain of function I should generate Lentivirus with the:
lenti dCAS-VP64_Blast, Retro, Gag/Pol ...
nucleofection with Lonza of suspension cells- low efficiency with 10 kb plasmid
nucleofection with Lonza of suspension cells- low efficiency with 10 kb plasmid
I have been working with CRIPSR-Cas system and using nucleofection method for plasmid delivery. I am co-transfecting with10 kb size CRISPR-Cas and 3kb size gRNA using Lonza amaxa 4D-X system. The cells are suspension B cells. Buffers for transfection are company SF buffer and also homemade buffers of which recipes obtained ...
DNA is edited but protein still remains, is this a problem?
DNA is edited but protein still remains, is this a problem?
A while back I performed a genome-wide CRISPR dropout drug screen in a leukemia cell line and found some pretty cool hits. I then followed up the screen with some validation experiments where I performed a reporter (RFP)-based competition assay that was assessed via flow cytometry. In my screen I used the Brunello library w...
CRISPR Gecko v2 library with lentiviral ready.
CRISPR Gecko v2 library with lentiviral ready.
Would it be possible to buy the CRISPR Gecko v2 library lentiviral ready to use directly on human cell lines? I would be interested if anyone can share their resources. We bought Gecko V2 from addgene but like to minimize the preparation error and troubleshooting.
Will appreciate hearing your thought.
Basic question about sgRNA designing
Basic question about sgRNA designing
Hi all!
I am very very new to the CRISPR/Cas technology and I will be needing it for my PhD project. Apologies from before if the questions I ask are very basic. I have been doing some reading and have two questions in mind -
1\. Do we need to choose any specific Cas9 in order to generate a stable Cas9 cell line? Where we...
CRISPR library transduced cell numbers I need to keep during passage after puromycin selection
CRISPR library transduced cell numbers I need to keep during passage after puromycin selection
I am a beginner of CRISPR pooled screen. I read the paper titled "Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening" (Julia Joung et al., 2017). In the paper, for a library size of 100,000 unique sgRNAs, we transduce 1.67 × 10^8 cells at an MOI of 0.3 at the start. After puromycin addition, I need...