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
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 ...
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...
Should I maintain Antibiotics (Blasticidin and Puromycin) Selection throughout the screen?
Should I maintain Antibiotics (Blasticidin and Puromycin) Selection throughout the screen?
I am currently working on a CRISPR screen using the Brunello Library, 2-vector system. The Cas9 portion contains Blasticidin selection and the sgRNA library has puromycin selection.
After infection and antibiotic selection (with both Blast and Puro), we did several rounds of FACS. However, after sorting, we found that thos...
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...
Are all gRNA expression systems compatible with CRISPRko/CRISPRi/CRISPRa?
Are all gRNA expression systems compatible with CRISPRko/CRISPRi/CRISPRa?
Assuming the (d)Cas9 is expressed separately, would one gRNA expression vector work for CRISPRko, CRISPRi, and CRISPRa?
Eg, can I use pLentiGuide for CRISPRko, CRISPRi, and CRISPRa assuming the cells have the appropriate (d)Cas9?
Calabrese(humanCRISPRa) Library_trouble in amplifying genomic DNA
Calabrese(humanCRISPRa) Library_trouble in amplifying genomic DNA
I just had an issue in amplifying the gemomic DNA from unsorted cells sample. Briefly, I did a sorting-based screen with the Calabrese library(human CRISPRa) and used the Ex-taq protocol mentioned in their paper , and failed in amplifying the genomic DNA from unsorted cells.
(1 round PCR, 28cycles)
Shown as below the PCR ...
Custom-made CRISPR pooled gRNA KO library_Lentiviral vectors
Custom-made CRISPR pooled gRNA KO library_Lentiviral vectors
I want to use a CRISPR library to knockout a certain number of human genes that I'm interested in.
Since I'm new in the field of genome editing, I would like to find a reliable company to generate for me the library in the form of ready-to-transduce lentiviral-vectors.
I am going to follow the Nature protocols 2017 of Jou...