The CasRx entry in FlyBase represents an RNA-guided ribonuclease which is derived from the naturally occurring type VI-D CRISPR ribonuclease encoded by the Ruminococcus flavefaciens XPD3002 Cas13d gene and in which the two HEPN ribonuclease domains present in the naturally occurring protein are functional. The exact sequence of the nuclease may differ depending on the particular transgene or modified endogenous locus being used, for example, the coding sequence may have been codon optimized for use in different species and a nuclear localization signal may have been added. Cas13d ribonucleases specifically target single-stranded RNA for cleavage. They are able to process a precursor CRISPR repeat array into mature guide RNAs (gRNAs), each of which contains a spacer sequence complementary to the target RNA, plus additional sequence required for binding to Cas13d. In the presence of the mature gRNA, Cas13d recognises and binds the complementary target RNA. This forms an active ternary complex in which the two HEPN ribonuclease domains of Cas13d act together to cleave the complementary target RNA. This cleavage has no apparent protospacer flanking sequence requirements (PMID:29551272, PMID:31186424). These properties mean that CasRx can potentially be used to target specific RNAs of interest for knockdown, by providing either an unprocessed guide RNA (pre-gRNA) array or a mature gRNA that targets the RNA of interest; use of a pre-gRNA array would allow multiple gRNAs that target different sequences of the same RNA or that target multiple RNAs to be supplied simultaneously. However, in bacterial cells and in vitro, cleavage of collateral or 'bystander' RNAs (that are not complementary to the gRNA) by the activated ternary complex is also seen, potentially limiting the use of CasRx for specific RNA targeting. When expressed in transgenic Drosophila (FBrf0246003), CasRx can produce a moderate on-target reduction in transcript levels, however, variable levels of off-target activity are also seen (it is not clear whether the off-target activity is due to guide RNA-specific effects and/or is due to collateral targeting of bystander RNAs). In addition, expression of CasRx in transgenic Drosophila is often toxic and results in unexpected lethality when expressed with a guide RNA, limiting its use in flies without further optimization (FBrf0246003). For a comparison of CasRx with other CRISPR-based RNA-targeting systems, see PMID:29940185 and PMID:30388409.