Easy multiple sequential CRISPR/Cas9 knockouts in cell lines using a Cre/LoxP re-cyclable vector
Dong, L.; Etemadi, N.; Vaux, D. L.
Show abstract
To easily generate cell lines lacking multiple proteins, we inserted Cre/Lox sites flanking the guide RNA, Cas9, and mCherry fluorescent protein coding regions of a CRISPR/Cas9 lentiviral vector. Cells bearing an inducible Cre recombinase construct can be transfected with the CRISPR/Cas9 lentiviral vector, and mCherry positive cells sorted by flow cytometry. Induction of Cre causes deletion of the guide RNA, Cas9, and mCherry genes, so that mCherry negative cells can be isolated. After confirming successful targeting of the gene, the cells can be re-infected with the same vector bearing a different guide RNA, and mCherry-positive cells sorted once more. In this way, multiple genes can be mutated sequentially using the same vector and selection marker, without persistent expression of the guide RNA or Cas9. We used this system to sequentially mutate two candidate genes, Bak1 and Bcl2, and generated lines that lacked expression of both proteins.
Matching journals
The top 4 journals account for 50% of the predicted probability mass.
Similar papers in this journal
- Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering 95%
- A versatile vector system for the fast generation of knock-in cell lines with CRISPR 94%
- A cellular stress response induced by the CRISPR/dCas9 activation system is not heritable through cell divisions 94%
Similar papers in this journal
- Synthetic Circuits Based On Split Cas9 To Detect Cellular Events 94%
- A versatile bulk electrotransfection protocol for mouse embryonic fibroblast and iPS cells 94%
- Tagging allows faithful tracing of expression and enhances biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin. 93%
Similar papers in this journal
Similar papers in this journal
- Functional analysis of promoters for driving long RNA transcripts in CAR T cells 94%
- A Simplified Function-First Method for the Discovery and Optimization of Bispecific Immune Engaging Antibodies 94%
- APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression 93%
Similar papers in this journal
- Potent programmable antiviral against dengue virus in primary human cells by Cas13b RNP with short spacer and delivery by virus-like particle 94%
- In vitro and In vivo Genetic Disease Modelling via NHEJ precise deletions using CRISPR/Cas9 93%
- Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus 92%
"Similar papers" are the closest papers from that journal in the model's embedding space. They show what the match is built on, but the ranking comes mostly from a classifier over the whole training set, not from these examples alone.