Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control

Abstract

Our approach aims to optimize postscreening target validation strategies using viral vector-driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE-VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.

Inhibition of histone deacetylation rescues phenotype in a mouse model of Birk-Barel intellectual disability syndrome

“…Viral constructs were purchased from Sirion Biotech (Martinsried, Germany). Generation and evaluation of shRNAmir sequence targeting the gene Kcnk9 were performed using SIRION's RNAiONE platform as previously described 38 . Ten different 21mer shRNAmir sequences against Kcnk9 were selected using algorithms listed in Fig.…”

The King Is Dead, Long Live the King! JBS Special Issue on Screening by RNAi and Precise Genome Editing Technologies

“…The major concern of RNAi is off-target effects, and a number of strategies have been developed to address this issue. Here, Christel et al 11 describe a postscreening strategy using overexpression of candidate RNAi hits to verify the on-target phenotype of the shRNA screen. Their method allows validating top candidates stemming from screens by quickly designing efficient shRNA vectors to replicate the phenotype and then revert it by expression of a silencing resistant version of the gene.…”