I am new to the use of CRISPR libraries and I would like some input regarding a problem we are having. I am. using the GeCKO v2 library according to the Joung et al. Nature protocols paper.
After library amplification we amplified the sgRNA targets by PCR using an equimolar mix of the primers foward of the paper and one of ...
Hello all
I recently amplified the human GeCKO v2 and SAM libraries following the Joung et.al. 2017 protocol. I used the count_spacers.py script with a few modifications to run with phyton 3 and obtain the following statistics.
GECKO-A ...
Chartreuse Mermaid
over 1 year ago
Guide design
I want to validate some genes of interest coming out from the
screening with the gecko v2 library.
I'd like to take the guides of the library to singular validate in my
target cells.
Normally I design the guide in the following way:
fwd: 5'-CACC- guide sequence- 3'
rev: 3'- AAAC- reverse guide sequence-5'
Then I do the ann...
Terracotta Zombie
over 1 year ago
KO validation in Gecko v2 library B
I'm validating the data obtained with the screening by testing a pool of 3 guides belonging to gecko V2 library B.
I want to confirm the KO using the sanger sequencing. In some guides I've obtained a single SNP, could it be that some of the guides you designed work by introducing SNPs?
Turquoise Ghoul
about 2 years ago
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.
I just generated the Gecko V2 human sgRNA plasmid library from addgene. When I ran the count_spacers.py as
python ../ScreeningProtocolsmanuscript/countspacers.py -f ZXGecko2AS1R1001.fastq -i humangeckov2librarya09mar2015.csv,
the statistics.txt looks like:
Number of perfect guide matches: 0
Number of nonperfect guide ma...
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...
Bronze Basilisk
almost 3 years ago
Positive screening and no hits with low FDR
We performed a positive screening with GECKO v2 full library and after screening and NGS, we could see none of the genes had a positive ranking with lower FDR. With only one Gene as a top hit, with lower FDR which we think as a result of positive screen selection.
Please suggest some inputs regarding this issue.
Thanks in a...
I'm planning to use a custom-made CRISPR knockout library to target approximately 300 human genes.
I am going to follow the Nature protocols 2017 of Joung et al. that I found really helpful !
My questions are:
Considering the number of genes that will be targeted, is it better to use 6 or 4 sgRNAs per gene (similarly t...
Recently, I have amplified Mouse CRISPR Knockout Pooled Library (GeCKO v2) as described in protocol. Because I didn't mix them in any process, I acquired two amplified half libraries. After that, I qualified these two half libraries by NGS sequencing, and decided to screen the entire library in further studies. However, sin...
Find your community. Ask questions. Science is better when we troubleshoot together.
Community Guidelines
Sci Find is a place for experimentation beyond the publication. We are a growing collaborative community of scientists and experimentalists from around the world who believe science is better when we work together.
- Help us keep Sci Find a safe space for productive and collaborative conversations. Please be kind and respect your fellow community members. If you see abusive or alarming content, please report it to admin@scifind.net, and our team will take appropriate actions.
- All information posted to this page is public and Open Access. Learn more about our commitment to Open Access by visiting our Docs.
- We encourage our community to have collaborative troubleshooting discussions, and to share tips and tricks, experimental methods, resources, and opportunities.
- You must be a member to create or engage with content. Sign Up to create your profile and join the community for free.
- Follow a guild and create a post. Learn more about guilds here.
- Post titles should be descriptive and include descriptive tags. Check out our tagging guidelines here.