The introduction of CRISPR/Cas9 technology has taken the functional genomics field by storm, allowing genomic alterations with high degree of precision and throughput, thanks to the availability of genome wide libraries. 

These libraries are either formatted as “pooled”, where guide RNAs, usually packed in lentiviral vectors, are introduced to the entire cell population and selected, or “arrayed”, where the single guide RNAs are introduced into individual populations in microtiter plates allowing the manipulation of a single gene per well. Both approaches present considerable challenges at various levels: in the case of arrayed screen, one of the bottlenecks is the simultaneous and efficient delivery of both the Cas9 protein and the gRNAs nucleic acid into the cells.

We will present the solutions we have developed to tackle the delivery problem in the context of image based phenotypic assays, to be deployed in future genome-wide campaigns. We will focus on the development and validation of procedures of transient transfection either using complexes of recombinant CRISPR/Cas9 and gRNA, or adopting an uncoupled approach where the Cas9 is delivered using the BACMAM system, followed by gRNAs transfection.