Shen et al (Nature Methods, 2017) explore and identify synthetic interactions among 73 cancer-associated genes. To perform their loss of function screen they combined CAS9-expressing cell lines with a sgRNA library of high titer lentiviral particles. Most of these gene interactions were subsequently validated by drug treatment. [Read more…]
Using CRISPR to knockout an essential gene
Genome editing technology enabled by CRISPR and TALEN has become mainstream. Most cell biology labs are engaged in projects to create custom cell lines with knock-outs and knock-ins, and companies such as GeneCopoeia even propose complete cell line generation services. Projects can involve transfection of mammalian expression constructs, TALEN pairs, or direct transfection of RNA.
When scientists want to make a stable knockout cell line, one of the first questions they should ask is whether or not the resulting cell line will be viable. Often a murine knockout mouse has been made and/or siRNA knockdown experiments have been performed in human cells, so experienced users have a good idea if a human knockout cell line will be viable. There are certainly some cases, however, where either the researcher knows or expects that a complete knockout will not be viable but wishes to make the knockout nonetheless. What is the best way to deal with all of the risk involved with starting an relatively expensive and time-consuming project like this that could end in failure? [Read more…]