I'm looking for a good selectable, lentiviral system to knock out genes in human cells. But so far too few people could share their experience on the available systems that convinced me of the best one.
Hey Patricia,
you should definitely look into the pLENTI-CRISPR-v2 construct. It is a chimeric lentiviral backbone containing gRNA and Cas9 cassettes, for simple Lenti virus delivery with subsequent Puro selection. I have used this construct in the past and it works very well. But, the efficacy/quality of your knockout depends on the guide RNA!
https://www.addgene.org/52961/
Good luck.
Hi Patricia,
I'm using a lenti system from Zhang's lab as well. http://www.ncbi.nlm.nih.gov/pubmed/25075903
It's very important to get a good gRNA for your constracts. There are some softwares available.
Good luck!!
Dear Patricia
I suggest you to use CRISPR-Fok1 instead of using lenti system. Tis approved that off-target effects of the CRISPR/Cas9 have correlation with the amount of Cas9 expression. Hence, applying a system which has the least off-target activity especially in human cells is crucial. Up today, the CRISPR-Fok1 has the highest efficiency and the least off-target activity in knocking-out approaches. You should work with pSQT1601 and pSQT1313 constructs.
Good luck,
Dear Rita
kindly see Dr. Joung's report. (2014) "Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing".
Good luck,
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research