RNA interference (RNAi) technology used for the functional analysis of Caenorhabditis elegans genes frequently leads to phenotypes with low penetrance or even proves completely ineffective. The methods previously developed to solve this problemwere built on mutant genetic backgrounds, such as those defective for rrf-3, in which endogenous RNAi pathways are overexpressed. These mutations, however, interferes with many other genetic pathways so that the detected phenotype cannot always be clearly linked to the RNAi-exposed gene. In addition, using RNAioverexpressing mutant backgrounds requires timeconsuming genetic crossing. Here, we present an improved RNAi vector that produces specific doublestranded RNA species only, and thereby significantly stronger phenotypes than the standard gene knockdown vector. The further advantage of the new RNAi vector is that the detected phenotype can be specifically linked to the gene silenced. We also created a new all-in-one C. Elegans Cas9 vector whose spacer sequence is much easier to replace. Both new vectors include a novel CRISPR/Cas9-based auto-cloning vector system rendering needless the use of restriction and ligase enzymes in generating DNA constructs. This novel, efficient RNAi and autocloning Cas9 systems can be easily adapted to any other genetic model.
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