Looking to overcome the limitations of RNAi-based genetic screens, researchers from the Wellcome Trust Sanger Institute recently reported on the use of the gene-editing method CRISPR to conduct genome-wide loss-of-function screens in mammalian cells.
Despite its robustness and wide adoption, RNAi often produces off-target effects and sometimes fails to suppress the expression of its target enough to enable observation of knockout phenotypes. Introducing mutations into mammalian haploid cells offers an alternative to RNAi, but the haploid state can only be stably maintained in a few cell lines, limiting its utility.
CRISPR, however, offers a solution to this issue. Short for clustered regularly interspaced short palindromic repeats, CRISPR works when a single-stranded RNA complementary to a specific DNA sequence is introduced into a cell and acts as a guide for a complex containing the nuclease Cas9, which cuts both strands of the target DNA region.
In their study, which was published in Nature Biotechnology in December, the scientists used a CRISPR/Cas system to introduce genome-wide targeted mutations in mouse embryonic stem cells.
They designed more than 87,000 guide RNAs (gRNAs) targeting more than 19,000 mouse protein-coding genes and used a lentiviral vector to express the 20 nucleotide-long gRNAs in stem cells that constitutively express Cas9.
They conducted two genetic screens using the gRNA libraries to look for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine, and identified 27 known and four previously unknown genes implicated in resistance.
According to Kosuke Yusa, the study's senior author, the CRISPR screening approach not only overcomes the problem of insufficient target knockdown and off-targeting associated with RNAi screening, but is also easily adoptable by any researcher.
The team's genome-wide library is available from Addgene, as are libraries made by other teams, he said in an email.
"All these libraries work based on almost exactly the same experimental procedures for shRNA libraries: generation of mutant cell libraries, screening, and post-screening analysis," which makes the method especially straightforward for those who have experience with RNAi screening, he added.