A handful of recent papers and preprints have highlighted some important considerations for using TRiP shRNA and sgRNA stocks.

First, the Weake lab at Purdue University discovered that TRiP stocks contain a previously uncharacterized loss-of-function sevenless allele (sev²¹) on the X-chromosome (PMID: 32550423). TRiP stocks containing sev²¹, including both RNAi and sgRNA lines, have been annotated on Flybase and at BDSC. The presence of the sev²¹ mutation will not generally affect the use of these stocks, as the X chromosome is typically segregated out or heterozygous during experiments.

Next, a preprint from the Stern lab at Rice University showed that the attP40 docking site, used in a large percentage of TRiP stocks, is an insertion mutation of the Nesprin-1 orthologue MSP300 (doi.org/10.1101/2022.05.14.491875). Some, but not all, attP40 insertions cause larval muscle nuclear clustering and lethality. Importantly, this is a recessive lethal phenotype, and we note that most of the time TRiP stocks are heterozygous during RNAi or CRISPR experiments. Further, more than 80% of TRiP attP40 insertions are, in fact, homozygous viable, indicating that more work needs to be done to clarify the degree that the reported lethality is due to the disruption of MSP300. For now, we suggest that users of TRiP stocks working on muscle pay particular attention to their controls and make sure that attP40 insertions remain heterozygous during experiments.

Finally, a preprint from the Uhlirova lab (doi.org/10.1101/2022.08.12.503771) reported that VALIUM10 TRiP RNAi lines can silence Gateway-based transgenes that are also present in experiments. Users should note that the effect only applies to the roughly 1800 VALIUM10 RNAi lines (~11% of all TRiP RNAi lines) not to VALIUM1, VALIUM20, VALIUM21, VALIUM22, or pNP lines. Also, as we noted in the original paper describing VALIUM10 (PMID: 19487563), any potential siRNAs derived from the additional sequences in VALIUM10 do not lead to sequence-specific off-target effects on endogenous genes, and so should not have any effect on the use of the lines for loss-of-function experiments.